JP6833812B2 - Promoter mutant - Google Patents

Description

The present invention is an isolated artificial promoter, which is a functional variant or derivative of the pG1 promoter regulated by a carbon source of the methanol-utilizing yeast (Pichia pastoris) identified by SEQ ID NO: 1. In, it is a promoter called pG1-x, which refers to an isolated artificial promoter characterized by a specific promoter element and characteristics.

background

The methanol-utilizing yeast (Pichia pastoris), a methylotrophic yeast (synonymous with the genus Komagataella), is a well-established protein-producing host. Numerous strain manipulation methods for methanol-utilizing yeast (P. pastoris) have improved productivity for a variety of products, and efforts have also been made to promoters for production. . Microb Cell Fact, 12: 5). The gene promoter is a key feature for expressing the gene of interest (GOI): transcription of RNA in the downstream (3') GOI is driven by the upstream (5') promoter sequence. To. RNA polymerase II (RNAPII) contributes to the transcription of mRNA in eukaryotes. The RNAPII promoter consists of a core promoter and several cis-activating DNA elements: a proximal promoter, an enhancer, a silencer, and a boundary / insulator element. Yeast core promoters are typically located in the vicinity of the major transcription initiation site (-75 / + 50 bp) and they are frequently inadequate TATA boxes (with a difference of 2 bases or less to the TATA consensus sequence). Contains and lacks promoter elements typically found in other organisms. Transcriptional regulation is mediated by cis-activating elements and corresponding regulatory proteins (transcription factors (TFs)) in response to different conditions.

For biotechnology applications, promoters that allow constitutive or regulatory / inducible gene expression are used. For production processes that utilize methanol-utilizing yeast (P. pastoris), it is convenient to apply a _{carbon source-dependent promoter, such as methanol-induced PAOX.} This allows the growth phase to be separated from the potentially cumbersome protein production phase. In recent years, a series of promoters that are also controlled by carbon sources but do not rely on methanol for induction have been reported (Prielhofer et al., 2013): These promoters are suppressed by excess glycerol and induced by restricted glucose. Share the characteristics of. The most potent of these promoters, pG1 (SEQ ID NO: 1), is fully induced with less than 0.05 g of glucose per liter; in nature, it regulates the expression of the high-affinity glucose transporter gene GTH1. The characteristics of glucose uptake depend on the presence of high and low affinity glucose transporters. The 17 hexose transport (HXT) genes (HXT1-17) in S. cerevisiae are expressed in response to glucose concentration, and only two HXT homologues are methanol-utilizing yeast (P. pastoris). Found within (PAS_chr1-4_0570 and PAS_chr2-1_0054, named PpHxt1 and PpHxt2). PpHxt1 has been identified as the major low-affinity transporter within the methanol-utilizing yeast (P. pastoris), whereas high-affinity glucose transport is associated with two other genes: PAS_chr3_0023 and PAS_chr1-. This is facilitated by 3_0011 (GTH1, a gene regulated by pG1; Prielhofer et al., 2013).

Saccharomyces cerevisiae features enormous glucose uptake and (fermentative) glucose metabolism, whereas P. pastoris has a low glucose uptake rate and respiratory glucose. Has metabolism. In addition, methanol-utilizing yeast (P. pastoris) is capable of consuming glucose at much lower extracellular concentrations than Saccharomyces cerevisiae (S. cerevisiae) (mM range in S. cerevisiae). versus a μM range in the methylotrophic yeast (P. pastoris), K _M of the high affinity transporters). Fundamental differences in glucose uptake behavior are also manifested in the transcriptional regulation of related genes and can also be seen in the evolution of the functions of transcriptional regulators such as PpAft1 and PpMxr1 (homologues of ScAdr1).

Studies on the promoter of P. pastoris, as well _{as random mutagenesis of P GAP , the promoter of PAOX1} and glyceraldehyde-3-phosphate dehydrogenase, have different activities compared to wild-type promoters. brought the library with the promoter variants that possess a modified induced behavior of P _AOX1, some important transcription factor binding site (TFBS) and identified as a result (WO2006 / 089329A2).

The pG1 promoter and fragments thereof are further described in WO2013 / 055051A1.

WO2014067926A1 discloses the expression of a protein of interest with the aid of a specific leader sequence. The leader was used with a variety of promoters. As an exemplary promoter, the pG1 promoter was used.

Struhl K. (Proceedings of the National Academia of Sciences of the United States of America, 1982, 78 (7): 4461-4465) describes a deletion mapping of the yeast his3 promoter region. Struhl K. Concludes that the TATA box sequence preceding most eukaryotic genes is not sufficient for the function of the wild-type promoter, and the yeast promoter is a simple site of interaction between RNA polymerase and DNA. It suggests that it is considered to be more complex.

Quandt et al. (Nucleic Acids Research, 1995, 23 (23) 4878-4884) describe a tool for detecting consensus matches in nucleotide sequence data and identifying regulatory motifs based on sequence data analysis. .. A library of consensus patterns was created and a software tool (MatInspector) was used to detect potential sequence matches.

It is an object of the present invention to provide improved regulated promoters with respect to regulation by carbon sources and promoter strength. It is a further objective to provide such a promoter for enhancing POI production and / or producing POI within a reduced time.

This objective is achieved by the claimed subject matter.

According to the present invention, isolated and / or artificial pG1-x, which is a functional variant of the pG1 promoter regulated by a carbon source of methanol-utilizing yeast (Pichia pastoris) identified by SEQ ID NO: 1. A promoter, wherein the pG1-x promoter comprises or comprises at least a portion of SEQ ID NO: 1 having a length of at least 293 bp and comprises the following promoter regions:
It features a) at least one core regulatory region comprising the nucleotide sequences of SEQ ID NO: 2 and SEQ ID NO: 3; and b) a non-core regulatory region that is any region within the pG1-x promoter sequence other than the core regulatory region. ,
The pG1-x promoter has at least one mutation in any of the promoter regions, at least 80% sequence identity in SEQ ID NO: 2 and SEQ ID NO: 3, and any other than SEQ ID NO: 2 or SEQ ID NO: 3. Includes at least 50% sequence identity in the region of;
The pG1-x promoter is characterized by the same or increased promoter intensity and induction ratio as compared to the pG1 promoter, in this case.
-The promoter intensity is increased by at least 1.1 times compared to the pG1 promoter and / or-the induction ratio is increased by at least 1.1 times compared to the pG1 promoter in the induced state.
The pG1-x promoter is provided.

Specifically, the pG1 promoter of the methanol-utilizing yeast (Pichia pastoris) identified by SEQ ID NO: 1 is any of SEQ ID NOs: 7, 8 or 9, and more specifically, the example herein. In SEQ ID NO: 9, which is used as a reference.

Specifically, the pG1-x promoter is any of the prior art promoters named pG1 (SEQ ID NO: 264), pG1a (SEQ ID NO: 265), pG1b (SEQ ID NO: 265) described in WO2013050551A1. 266), pG1c (SEQ ID NO: 267), pG1d (SEQ ID NO: 268), pG1e (SEQ ID NO: 269), or pG1f (SEQ ID NO: 270).

According to a specific embodiment, the pG1-x promoter according to the present invention is:
-In the induced state, at least 1.1 times, or at least 1.2 times, or at least 1.3 times, or at least 1.4 times, or at least 1.5 times, or at least 1. 6 times, or at least 1.7 times, or at least 1.8 times, or at least 1.9 times, or at least 2 times, or at least 2.1 times, or at least 2.2 times, or at least 2.3 times, Or at least 2.4 times, or at least 2.5 times, or at least 2.6 times, or at least 2.7 times, or at least 2.8 times increased promoter strength, or at least 2.9 times, or at least 3 times. Or at least 3.3 times, or at least 3.5 times, or at least 3.8 times, or at least 4 times, or at least 4.5 times, or at least 5 times, or at least 5.5 times, or at least 6 times. Great promoter strength and
• Ability regulated by a carbon source, characterized by an ability that is determined by the same or greater induction ratio as compared to the induction ratio achieved by the pG1 promoter. A possible promoter.

According to a more specific embodiment, the pG1-x promoter according to the present invention is:
• In the induced state, with the same or greater promoter strength compared to the pG1 promoter. • At least 1. The ability to be regulated by the carbon source and compared to the induction ratio achieved by the pG1 promoter. 1x, or at least 1.2x, or at least 1.3x, or at least 1.4x, or at least 1.5x, or at least 1.6x, or at least 1.7x, or at least 1.8 Double, or at least 1.9 times, or at least 2 times, or at least 2.1 times, or at least 2.2 times, or at least 2.3 times, or at least 2.4 times, or at least 2.5 times, or An induction ratio that is at least 2.6 times, or at least 2.7 times, or at least 2.8 times higher, or at least 2.9 times, or at least 3 times, or at least 3.3 times, or at least 3.5 times. Alternatively, it is characterized by an ability determined by an induction ratio that is increased by at least 3.8 times, or at least 4 times, or at least 4.5 times, or at least 5 times, or at least 5.5 times, or at least 6 times. It is a promoter that can be regulated by the carbon source.

According to a more specific embodiment, the pG1-x promoter according to the present invention is:
-In the induced state, at least 1.1 times, or at least 1.2 times, or at least 1.3 times, or at least 1.4 times, or at least 1.5 times, or at least 1. 6 times, or at least 1.7 times, or at least 1.8 times, or at least 1.9 times, or at least 2 times, or at least 2.1 times, or at least 2.2 times, or at least 2.3 times, Or at least 2.4 times, or at least 2.5 times, or at least 2.6 times, or at least 2.7 times, or at least 2.8 times increased promoter strength, or at least 2.9 times, or at least 3 times. Or at least 3.3 times, or at least 3.5 times, or at least 3.8 times, or at least 4 times, or at least 4.5 times, or at least 5 times, or at least 5.5 times, or at least 6 times. Great promoter strength and
• Ability regulated by the carbon source, at least 1.1-fold, or at least 1.2-fold, or at least 1.3-fold, or at least 1. times the induction ratio achieved by the pG1 promoter. 4 times, or at least 1.5 times, or at least 1.6 times, or at least 1.7 times, or at least 1.8 times, or at least 1.9 times, or at least 2 times, or at least 2.1 times, Or at least 2.2 times, or at least 2.3 times, or at least 2.4 times, or at least 2.5 times, or at least 2.6 times, or at least 2.7 times, or at least 2.8 times. Induction ratio, or at least 2.9 times, or at least 3 times, or at least 3.3 times, or at least 3.5 times, or at least 3.8 times, or at least 4 times, or at least 4.5 times, or at least A carbon source-regulated promoter characterized by an ability determined by an induction ratio that is increased 5-fold, or at least 5.5-fold, or at least 6-fold.

Specifically, the promoter strength comprises a protein of interest (POI) compared to the pG1 promoter, eg, a model protein (eg, eg, enhanced GFP eGFP, Gene Bank accession number: U57607). It is determined by the expression level and / or transcription rate of GFP), which is a green fluorescent protein. Specifically, the promoter strength of pG1-x is at least 1.2 times, or at least 1.3 times, or at least 1.4 times, or 1.5 times, or at least 1 times that of the pG1 promoter, for example. 1.6 times, or at least 1.7 times, or at least 1.8 times, or at least 1.9 times, or at least 2 times, or at least 2.1 times, or at least 2.2 times, or at least 2.3 times Or at least 2.4 times, or at least 2.5 times, or at least 2.6 times, or at least 2.7 times, or at least 2.8 times, or at least 2.9 times, or at least 3 times, Or at least 3.5 times, or at least 4 times, or at least 4.5 times, or at least 5 times, or at least 5.5 times, or at least 6 times, or at least 6.5 times, or at least 7 times, or at least. It has increased by 7.5 times, or at least 8 times, or at least 8.5 times, or at least 9 times, or at least 9.5 times, or at least 10 times.

Here, the pG1 promoter may be used as a criterion or control for determining improvement in promoter function. Such a control pG1 promoter may be used in parallel control experiments using the same host cell and expression system, or as an internal control in the same host cell culture. Such control experiments that qualify the promoter function relative to the pG1 promoter preferably express a model protein, such as GFP or eGFP, in a methanol-utilizing yeast (P. pastoris) host cell culture. It is carried out in recombinant methanol-utilizing yeast (P. pastoris).

Induction by the pG1-x promoter specifically refers to the induction of transcription, which specifically comprises further translation and arbitrary expression of said POI.

The transcription rate is determined as a measure of promoter strength and specifically refers to the amount of transcript obtained when the promoter is fully induced.

The transcription rate may be determined, for example, by the transcription intensity in a fully induced state obtained under chemostat culture conditions with restricted glucose and may be expressed in comparison with the transcription rate of the pG1 promoter.

Preferably, the transcriptional analysis is quantitative or semi-quantitative, preferably utilizing qRT-PCR, DNA microarray, RNA sequencing, and transcriptome analysis.

Promoter strength compared to pG1 promoter strength can be determined by the following standard assay: under the control of the test promoter, a methanol-utilizing yeast (P. pastoris) strain expressing eGFP, per well. 2 mL of the culture is screened at 25 ° C. in a deep 24-well plate while shaking at 280 rpm. Glucose feed beads (6 mm, Kuhner, CH) are used to create glucose-restricted growth conditions. Cells are analyzed for expression of eGFP in the induced state (YP + 1 feed beads over 20-28 hours).

If the culture conditions result in near maximum induction, for example, at a glucose concentration of less than 0.4 g per liter, preferably less than 0.04 g per liter, specifically less than 0.02 g per liter, the promoter. It is considered that the suppression was released and the promoter was completely induced. The fully induced promoter is preferably at least 20%, more preferably at least 30%, 40%, 50%, 60%, 70%, 80%, 90% transcription compared to the native pGAP promoter. It indicates the rate and indicates a transcription rate of at least 100%, or even greater, at least 150% or at least 200%. The transcription rate may be determined, for example, by the amount of reporter gene, eg, eGFP transcript, when the clone is cultured in a liquid culture, for example, as described in the Examples section below. Alternatively, the transcription rate may be determined by the transfer intensity on the microarray, in which case the microarray data is large compared to the control in the fully induced state and the difference in the expression level between the suppressed state and the unsuppressed state. Indicates signal intensity.

Specifically, the native pGAP promoter is a promoter that is endogenous or homologous to eukaryotic cells that can be used as a host cell to determine POI expression and is used as a standard or reference promoter for comparison purposes. ..

For example, as the native pGAP promoter of P. pastoris is used to control the expression of GAPDH in P. pastoris, for example, the sequence shown in FIG. It is an unmodified endogenous promoter sequence in Pichia pastoris having a native pGAP promoter sequence (SEQ ID NO: 260) of P. pastoris (GS115). When methanol-utilizing yeast (P. pastoris) is used as a host for making POI according to the present invention, the transcriptional strength or rate of the pG1-x promoter according to the present invention can be determined by such methanol-utilizing yeast (P. pastoris). Compared to the native pGAP promoter of yeast) and / or compared to the native pG1 promoter.

As another example, the native pGAP promoter of S. cerevisiae is used within S. cerevisiae to control GAPDH expression within S. cerevisiae. , An unmodified endogenous promoter sequence. When S. cerevisiae is used as a host for making POI, the transcriptional intensity or rate of the pG1-x promoter is compared to the native pGAP promoter of such S. cerevisiae.

Therefore, the relative transcriptional strength or rate of promoters according to the invention is typically compared to the native pGAP promoter of cells of the same species or strain used as a host for making POI.

The induction ratio is a key parameter in determining the regulation of the pG1-x promoter, comparing the promoter activity or intensity in the inducing state with the promoter activity or intensity in the inhibitory state. For example, the expression level and / or transcription rate of a model protein (eg, GFP or eGFP) in a repressed state is determined when suppressed by excess glycerol, and the expression level and / or transcription rate of the model protein is a limitation of glucose feeding. It is determined by the induction state at the time of induction by.

Specifically, the induction ratio is determined by the ratio of expression levels (eg, GFP or eGFP) in the induced state as opposed to the suppressed state. The induction ratio of the pG1-x promoter is specifically the same or greater than that of the pG1 promoter. In specific cases, the induction ratio is at least 2-fold, or at least 3-fold, or at least 4-fold, at least 5-fold, or at least 6-fold, or at least 7-fold, at least 8-fold, or at least as compared to the pG1 promoter. It has increased by at least 9 times, or at least 10 times.

The induction ratio compared to the pG1 promoter intensity can be determined by the following standard assay: under the control of the test promoter, a methanol-utilizing yeast (P. pastoris) strain expressing eGFP per well. 2 mL of the culture is screened at 25 ° C. in a deep 24-well plate while shaking at 280 rpm. Glucose feed beads (6 mm, Kuhner, CH) are used to create glucose-restricted growth conditions. Cells are analyzed for eGFP expression during inhibition (YP + 1% glycerol, exponential growth phase) and induction (YP + 1 feed beads over 20-28 hours).

Specifically, the pG1-x promoter is at least 2.5-fold, or at least 3-fold, or at least 4-fold, at least 5-fold, or at least 6-fold, or at least 7-fold in the de-suppressed (induced) state. Has at least 8-fold, or at least 9-fold, or at least 10-fold promoter activity or intensity (eg, transcriptional activity or transcriptional intensity).

Specifically, when the core regulatory region incorporates the nucleotide sequences of SEQ ID NO: 2 and SEQ ID NO: 3, the sequences of SEQ ID NOs: 2 and 3 are placed in the pG1-x promoter sequence in any order, preferably with each other. In close proximity, for example, between the sequence of SEQ ID NO: 2 and the sequence of SEQ ID NO: 3 is meant to be included as 10, 20, 50, or 100 bp or less.

Specifically, SEQ ID NO: 2 and / or SEQ ID NO: 3 contains one or more transcription factor binding sites (TFBS).

Specifically, the nucleotide sequences of SEQ ID NO: 2 and SEQ ID NO: 3 include TFBS or at least a portion thereof, each or both of which sequences are considered to be recognized and functional by their respective transcription factors. Represent. Such nucleotide sequences of SEQ ID NO: 2 or SEQ ID NO: 3 (or functional variants thereof) are considered essential and, in unmodified form, or at least 80% of the sequences within the pG1-x promoter. It is incorporated as its functional variant with identity, or at least 85%, or at least 90%, or at least 95%, 100% or less sequence identity.

Specifically, the pG1-x promoter is a nucleotide sequence other than SEQ ID NO: 2 and SEQ ID NO: 3, with at least 50% sequence identity to the corresponding region within the pG1 promoter, specifically the core regulatory region or Includes nucleotide sequences having at least 60%, or at least 70%, or at least 80%, or at least 90% sequence identity in the non-core regulatory region. Specifically, the nucleotide sequence within the core regulatory region, which is any nucleotide sequence other than SEQ ID NO: 2 and SEQ ID NO: 3, is at least 60%, or at least 70%, or at least the corresponding region within the pG1 promoter. It has 80%, or at least 90%, or at least 95%, or at least 98% sequence identity. Specifically, the nucleotide sequence within the non-core regulatory region can have less than 90%, less than 80%, less than 70%, or less than 60% sequence identity to the corresponding region within the pG1 promoter.

Specifically, the core regulatory region comprises the nucleotide sequence of SEQ ID NO: 4, or a functional variant thereof comprising TFBS, preferably at least 80%, or at least 90%, or at least 95%, or at least 98% of the sequence. Includes or consists of functional variants with identity.

Specifically, the core regulatory region is sequenced with SEQ ID NO: 5, or a functional variant thereof, including TFBS, preferably at least 80%, or at least 90%, or at least 95%, or at least 98% sequence identity. Incorporate into the major regulatory regions represented by the associated functional variants.

Specifically, one or more TFBSs are TFBSs for any of the transcription factors selected from the group consisting of Rgt1, Cat8-1, and Cat8-2.

Specifically, TFBS is recognized by the transcription factors Rgt1 and / or Cat8-1 and / or Cat8-2. TFBS, which can fluctuate for the same factors, is characterized by a particular consensus sequence. Specific transcription factors are specified as follows.

Rgt1 is a glucose-responsive transcriptional activator and suppressor that regulates the expression of several glucose transporter (HXT) genes. Rgt1 of methanol-utilizing yeast (P. pastoris) is characterized by the amino acid sequence of SEQ ID NO: 261 (FIG. 7).

Cat8-1 and Cat8-2 are zinc cluster transcriptional activators that bind to carbon source response elements required for desuppression of various genes under non-fermentative growth conditions. Cat8-1 and Cat8-2 of methanol-utilizing yeast (P. pastoris) are characterized by the amino acid sequences of SEQ ID NOs: 262 and 263, respectively (FIG. 7).

Specifically, the core regulatory region comprises the deletion of one or more nucleotides between the nucleotide sequence of SEQ ID NO: 2 and the nucleotide sequence of SEQ ID NO: 3. The deletion may be one or more point mutations, one, two, three, four, five, six, located between SEQ ID NO: 2 and SEQ ID NO: 3. Refers to all 7, 8, or 9 nucleotides.

Specifically, the core regulatory region comprises the insertion of one or more nucleotides between the nucleotide sequence of SEQ ID NO: 2 and the nucleotide sequence of SEQ ID NO: 3. The insertion may be one or more point mutations and are located between SEQ ID NO: 2 and SEQ ID NO: 3, at least one, two, three, four, five, six, Refers to 7, 8, 9, or 10 nucleotides.

Specifically, the core regulatory region comprises the substitution of one or more nucleotides between the nucleotide sequence of SEQ ID NO: 2 and the nucleotide sequence of SEQ ID NO: 3. The substitution may be one or more point mutations, one, two, three, four, five, six, seven located between SEQ ID NO: 2 and SEQ ID NO: 3. Refers to all nine, eight, or nine nucleotides.

Any combination of specific deletions, insertions, or substitutions may be used to obtain the pG1-x promoter.

According to specific aspects, the pG1-x promoter comprises at least two copies of the core or major regulatory region, which is the original core regulatory region or functional variant, containing at least one mutation. .. Specifically, the pG1-x promoter may include at least two, three, or four copies of the core regulatory region and / or at least two, three, or four copies of the major regulatory region. ..

According to another specific aspect, the pG1-x promoter is at least two, three, or four of one or more TFBSs selected from the group consisting of Rgt1, Cat8-1, and Cat8-2. Includes 5, 6, 7, or 8 copies.

Specifically, the pG1-x promoter is preferably located at the 5'end of the pG1 sequence, leaving at least 280 nucleotides of the functional variant of the 3'or region of the pG1 sequence. It is a functional variant of the improved pG1 promoter that contains a deletion of one or more nucleotides.

According to a specific embodiment, the pG1-x promoter comprises at least one or at least two T motifs identified by any of SEQ ID NOs: 12-29. Specifically, the T motif
a) A sequence of continuous T (thymine), where T _n or (T) _n [in the sequence, preferably n = 13-20, preferably the T motif is T14, T15, or T16];
b) A sequence characterized by a sequence of continuous T (thymine) following A (adenine) at the first position, where ATn or A (T) _n [preferably n = 13 in the sequence. ~ 20, optionally preferably n = 13-22];
c) A sequence characterized by a sequence of continuous T (thymine) following T (thymine) in the first position and A (adenine) in the second position, where TATn or TA (T) _n. [Preferably n = 13 to 20 in the sequence].
d) a sequence wherein A (adenine) in the sequence and the last position of the continuous T (thymine), wherein, TnA or (T) _n A [in sequence, preferably, n = 13 to 20] Sequence called;
e) A sequence characterized by A (adenin) at the penultimate position and T (timine) at the last position following the sequence of continuous T (timine), where TnAT or (T). _n A sequence referred to as AT [preferably n = 13-20]; or f) A (adenine) replaced with T (timine), c) or e) sequence, wherein. TTTn or TnTT or T (A / T) Tn or T (A / T) (T) _n , or Tn (A / T) T or (T) _n (A / T) T [preferably n in the sequence. = 13-20], eg, any of the sequences resulting in a T motif consisting of the sequence _{(T) n [in the sequence, n = 15-22].}

For example, promoter sequences, [in the sequence, n = 13~20] TA (T ) n motif and one T motifs is, another is a (T) _n motifs [in sequence, n = 13 to 22] T Any of the T motifs specified under a) to f) above may be combined in one promoter sequence so as to include the motif.

The T-motif is optionally, with one or more "A" s (eg, one, two, or three adenines) at the 3'and / or 5'ends of the T-motif, and optionally further. Includes an extension such as with an "T" (eg, one, two, or three thymines), which extension is also referred to herein as an extended T motif.

Here, the term "T-motif" shall always include an extended or non-extended T-motif, and thus the term specifically includes both non-extended and extended T-motifs. Including.

Specifically, the T motif comprises or consists of a nucleotide sequence that is any of SEQ ID NOs: 12-29. Any one, two or more of the T motifs may be incorporated into the pG1-x promoter with extension of the motif or may be incorporated into the pG1-x promoter without extension of the motif.

According to one specific aspect, the extension of the T motif is an extension of the "TA" sequence at its 5'end to obtain a "TAT" 5'end.

According to another specific aspect, the extension of the T motif is an extension of the "TAA" sequence at its 5'end, an extension to obtain the "TAAT" 5'end.

According to another specific aspect, the extension of the T motif is an extension of the "AT" sequence at its 3'end, an extension to obtain the "TAT" 3'end.

According to another specific aspect, the extension of the T motif is an extension of the "AAT" sequence at its 3'end, an extension to obtain the "TAAT" 3'end.

According to a specific aspect, the T motif is located upstream of the core regulatory region and optionally upstream of the major regulatory region.

According to another specific aspect, the T motif is located downstream of the core regulatory region and optionally downstream of the major regulatory region.

Specifically, the pG1-x promoter comprises a 3'-terminal nucleotide sequence that incorporates at least a portion of the translation initiation site. The translation initiation site is specifically known as a Kozak consensus sequence in eukaryotes and is a sequence suitable for supporting gene expression.

Specifically, the translation start site is
a) Derived from the pG1 promoter and consisting of or containing the nucleotide sequence of SEQ ID NO: 6 or a functional variant thereof with at least 90% sequence identity; or b) Pichia pastoris It is derived from any other promoter, or its functional variant with at least 90% sequence identity.

An exemplary alternative 3'end promoter region, which may be used in place of the 3'end region of the pG1 promoter or in place of the nucleotide sequence of SEQ ID NO: 6, is, for example, the following promoters. : Derived from any of pAOX1, pAOX2, pDAS1, pDAS2, pFLD, pGAP, or pTEF2.

According to a specific embodiment, the promoter has a length of 2000 bp or less. The specific pG1-x promoter has a shorter length than the pG1 promoter, with, for example, a length of at least 293 bp or 300 bp, or at least 328 bp, or at least 350 bp, or at least 400 bp, or at least 500 bp.

Specifically, the pG1-x promoter may include sequences derived from fragments of the pG1 promoter. According to specific aspects, the pG1-x promoter is a variant or derivative of the parent fragment of pG1 and is at least the 3'side region of SEQ ID NO: 1 and at least 50% or 60 of the pG1 sequence. %, Or 70%, or 80%, or at least 90% of the variant or derivative.

Specifically, the nucleotide sequence of pG1-x comprises a deletion in the 5'end region or a deletion in the 5'end region, eg, a cleavage of the nucleotide sequence at the 5'end, and varies from 3'end to 5'. 'Specific lengths with a range of edges, eg, at least 293 bp or 300 bp, or at least 328 bp, or at least 350 bp, or at least 400 bp, or at least 500 bp to at least 1, or at least 10, or at least. Derived from the nucleotide sequence of the pG1 promoter to obtain the length of the nucleotide sequence of the length of the pG1 promoter fragment containing a 100 bp deletion.

However, the promoter length can also be increased to obtain, for example, a length greater than the length of the pG1 promoter, specifically 1500 bp or less, or 2000 bp or less. Specifically, the length may be any of the range: 293 bp to 1500 bp, 293 bp to 2000 bp, 328 bp to 1500 bp, or 328 to 2000 bp.

According to specific aspects, the invention provides an isolated and / or artificial pG1-x promoter, which
a) Any of SEQ ID NOs: 37-44, preferably SEQ ID NOs: 45-76;
b) Any of SEQ ID NOs: 77-80, preferably SEQ ID NOs: 81-112;
c) Any of SEQ ID NOs: 113-114, preferably SEQ ID NOs: 115-130;
d) Any of SEQ ID NOs: 131-132, preferably SEQ ID NOs: 133-148;
e) Any of SEQ ID NOs: 149-150, preferably SEQ ID NOs: 151-166;
f) Any of SEQ ID NOs: 167 to 168, preferably SEQ ID NOs: 169 to 184;
g) Any of SEQ ID NOs: 185 to 186, preferably SEQ ID NOs: 187 to 202;
h) Any of SEQ ID NOs: 203-204, preferably SEQ ID NOs: 205-220;
i) Any of SEQ ID NOs: 221 to 222, preferably SEQ ID NOs: 223 to 238;
j) SEQ ID NOs: 239-240, preferably any of SEQ ID NOs: 241-256; and k) SEQ ID NOs: 32-36 or SEQ ID NOs: 257-259;
Containing or consisting of nucleotide sequences selected from the group consisting of any of the following;
Or l) is the pG1-x promoter, which is a functional variant of any of the above a) to k), preferably the pG1-x promoter is the same as compared to the pG1 promoter? Or characterized by increased promoter strength and induction ratio, in this case
-The promoter strength is increased by at least 1.1 times as compared with the pG1 promoter in the induced state, and / or-the induction ratio is increased by at least 1.1 times as compared with the pG1 promoter.

Such a functional variant of the pG1-x promoters a) to k) above is preferably any of the specific features described for the functional variant of the pG1 promoter described herein. It is characterized by.

Specifically, any of the functional variants of the pG1-x promoters a) to k) above, preferably any of the functional variants of SEQ ID NOs: 45-76, have the following characteristics:
a) The sequence contains a deletion of one or more nucleotides at the 5'end of the promoter sequence, preferably at least one of the functional variants of the 3'or region of the promoter sequence. A functional variant of any of the promoter sequences of the pG1-x promoters a) to k) above, which leaves 280 nucleotides, preferably 50, 100, 150, 200, 250, of the promoter sequence. Or a functional variant containing a 5'deletion of 300 nucleotides, including a 5'deletion up to, but not including, the major regulatory region, combined with any sequence downstream or 3'side of the major regulatory region. If there is more than one major regulatory region, then the 5'end deletion of the promoter sequence extends to the first or most 5'side major regulatory region but does not include it;
b) The sequence comprises one or more TFBSs, preferably the TFBS is a TFBS for any of the transcription factors selected from the group consisting of Rgt1, Cat8-1, and Cat8-2;
c) The core regulatory region comprises a nucleotide sequence of SEQ ID NO: 4, or a functional variant thereof comprising one or more TFBSs, preferably a functional variant with at least 80% sequence identity;
d) The core regulatory region is incorporated into a major regulatory region represented by a functional variant thereof, including SEQ ID NO: 5, or TFBS, preferably a functional variant with at least 80% sequence identity. thing;
e) The core regulatory region contains a deletion of one or more nucleotides between the nucleotide sequence of SEQ ID NO: 2 and the nucleotide sequence of SEQ ID NO: 3;
f) The sequence contains at least two copies of the core or major regulatory regions;
g) The sequence further comprises at least one or at least two T motifs identified by any of SEQ ID NOs: 12-29, preferably the T motifs are located upstream or downstream of the core regulatory region. , Optionally located upstream or downstream of the major regulatory region;
h) The sequence comprises a 3'-terminal nucleotide sequence containing at least a portion of the translation initiation site;
i) The sequence is characterized by one or more of extending to a length of 2000 bp or less.

The present invention also provides an isolated form of the pG1-x promoter.

Specifically, an isolated pG1-x promoter nucleic acid is provided that comprises the pG1-x promoter described herein, or a nucleic acid comprising a complementary sequence. Specifically, the complementary sequence is a sequence that hybridizes to the pG1-x promoter under strict conditions.

Specifically, the nucleic acid is operably linked to a nucleotide sequence encoding a protein (POI) of interest, which naturally does not associate with a nucleotide sequence encoding a POI. The POI is specifically a heterologous polypeptide or protein.

Specifically, the nucleotide sequence further comprises a nucleotide sequence encoding a signal peptide that allows the secretion of POI, preferably the nucleotide sequence encoding the signal peptide is flanked by the 5'end of the nucleotide sequence encoding POI. And are placed.

Specifically, the signal peptides include a signal sequence derived from the sprouting yeast (S. cerevisiae) alpha-zygote factor prepropeptide, and the acidic phosphatase gene (PHO1) and extracellular protein X (PHO1) of methanol-utilizing yeast (P. pastoris). Selected from the group consisting of signal peptides derived from EPX1) (Heiss, S., V. Puxbaum, C. Grubber, F. Altmann, D. Mattanovich, and B. Gasser (2015), Multistep protein processing of the secretion. of the extracellular protein Epx1 in Pichia pastoris and impedances on protein localization, Microbiology).

Specifically, the POI is a eukaryotic protein, preferably a mammalian protein.

In a specific case, the POI is a multimeric protein, specifically a dimer or tetramer.

According to specific embodiments, the POI is preferably an antibody or fragment thereof, enzymes and peptides, protein antibiotics, toxin fusion proteins, carbohydrate-protein conjugates, structural proteins, regulatory proteins, vaccines and vaccine-like proteins or particles. , Process enzymes, growth factors, hormones, and metabolites of cytokines, or POIs, specifically cells of recombinant cell cultures expressing the gene of interest under transcriptional control of the promoters of the invention. Heterologous proteins selected from therapeutic proteins, including metabolites.

Specific POIs are antigen-binding molecules, such as antibodies or fragments thereof. Specific POIs include antibodies such as monoclonal antibodies (mAbs), immunoglobulins (Ig: immunoglobulin) or immunoglobulin class G (IgG: immunoglobulin class G), heavy chain antibodies (HcAbs), or fragments thereof, such as , Fragment-antigen binding (Fab), Fd, single chain variable fragment (scFv), or engineered variants thereof, such as Fv dimer (diabody), Fv trimer (triaboyl). , Fv tetramers, or minibodies, and single domain antibodies, such as VH or VHH or V-NAR. Additional antigen-binding molecules may be selected from (alternative) scaffold proteins, such as engineered Kunitz domains, adnectins, affibodies, anticarins, and DARPins. The term "scaffold" describes a compact, stably folded, polyhedral protein group (different in size, structure, and origin) that is used as a starting point for the production of antigen-binding molecules. Inspired by the structure-function relationship of antibodies (immunoglobulins), such alternative protein scaffolds can be remodeled for close and specific recognition of a given (bio) molecular target. Provides a robust and conservative structural framework that supports.

According to specific embodiments, the fermented product is produced using POI, metabolites, or derivatives thereof.

The invention further provides an expression construct containing the nucleic acids described herein, preferably a self-replicating vector or self-replicating plasmid, or an expression construct that is a vector or plasmid that integrates into the chromosomal DNA of a host cell.

Specifically, the expression construct comprises said promoter operably linked to a nucleotide sequence encoding POI under transcriptional control of the pG1-x promoter, which is not naturally associated with the coding sequence of POI. Specifically, the expression construct is a vector.

The invention further comprises recombinant host cells containing the expression constructs described herein, preferably eukaryotic cells such as mammalian cells, insect cells, yeast cells, filamentous fungal cells, or plant cells, preferably yeast. Recombinant host cells, which are cells or filamentous fungal cells, more preferably yeast cells of the genus Saccharomyces or Pichia, are also provided.

Specifically, the yeasts are Pichia, Candida, Torulopsis, Arxula, Hansenula, Yarrowia, Kluyveromyces, genus Pichia, genus Candida, genus Torulopsis, genus Arxula, genus Hansenula It is selected from the genus, the genus Saccharomyces, the genus Candadaella, preferably the group consisting of methylotrophic yeast.

Specifically preferred yeasts are methanol-utilized yeast (Pichia pastoris), Komagataella pastoris, K. et al. K. phaffii, or K. Any of K. pseudopastoris, eg, a methanol-utilizing yeast (P. pastoris) strain, CBS704, CBS2612, CBS7435, CBS9173-9189, DSMZ70877, X-33, GS115, KM71, and SMD1168. Is it?

According to specific aspects, the recombinant host cell comprises multiple copies of the nucleic acid sequence and / or multiple copies of the expression construct. For example, recombinant cells include 2 copies, 3 copies, 4 copies or more (gene copy number: GCN (gene copy number)).

The present invention also provides stable cultures of the recombinant host cells described herein.

According to a specific embodiment, in the presence of a surplus carbon source, cells with a higher specific growth rate compared to restricted carbon source conditions are recruited.

The present invention further comprises a method of producing POI by culturing the recombinant host cell line described herein.
a) In the step of culturing the cell line under the conditions for expressing the POI,
b) Also provided is a method comprising the step of recovering POI.

Specifically, the method is performed under transcriptional control by a carbon source regulated pG1-x promoter, in which case the pG1-x promoter has improved promoter strength compared to the pG1 promoter. And have at least one of the adjustable features.

According to a specific embodiment, the cell line is cultured under batch, fed-batch, or continuous culture conditions and / or in a medium containing a restricted carbon substrate.

Specifically, the culture is carried out in the bioreactor with a batching period as the first step followed by a fed-batch or continuous culturing period as the second step.

Specifically, the host cell is in a high growth rate phase (eg, at least 50%, or at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or maximum. Growing in carbon-rich media (up to growth rate), POIs are low growth rate periods (eg, less than 90% of maximum growth rate, preferably less than 80%, less than 70%, less than 60%, 50). Less than%, or less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.4%, 0. Less than 3%, or less than 0.2%), preferably made by limited minimal medium feeding, eg, limiting the carbon source.

Specifically, POIs, for example, allow cell lines to grow below the maximum growth rate, typically less than 90%, preferably less than 80%, less than 70%, less than 60% of the maximum growth rate of cells. Less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.4%, 0. It is expressed under growth-restricted conditions by culturing at less than 3% or less than 0.2%. Typically, the maximum growth rate is determined individually for a particular host cell.

Specifically, the culture method is
a) First step of suppressing the pG1-x promoter by using a basal carbon source and subsequent b) No supplemental carbon source or a limited amount of supplemental carbon source This includes a second step of inducing the production of POI by unsuppressing or inducing the pG1-x promoter.

Specifically, a batch period is performed until the cell line consumes the basal carbon source initially added to the cell culture. Dissolved oxygen (DO) spike methods can be used to determine basic carbon source consumption during batch periods.

According to a specific embodiment, the batch phase is characterized by a sustained decrease in the oxygen partial pressure (pO2) signal and the end point of the batch phase is characterized by an increase in pO2. Typically, in the batch period, while consuming the basal carbon source, without adding additional carbon sources, as is typical for the batch period, the oxygen partial pressure (pO2) signal is, for example, 65%. It will decrease continuously, down to, for example, 30%. After consuming the basal carbon source, pO2 can increase, for example, above 30%, for example above 65%, so switch to a fed-batch system using feed medium to limit the carbon source. Below, the appropriate time points for adding additional carbon sources are indicated.

Specifically, during the batch period, pO2 is reduced to less than 65% or low saturation, followed by an increase or high saturation of more than 65% at the end of the batch. Specifically, the batch period until the increase in oxygen partial pressure (pO2) signal is saturated above 65%, specifically above 70%, 75%, 80%, or 85%. carry out.

Specifically, the batch period is carried out for about 20 to 36 hours.

The term "about" with respect to culture time shall mean ± 5% or ± 10%.

For example, a specific batch implementation time of about 20-36 hours means a duration of 18-39.6 hours, specifically 19-37.8 hours.

According to a specific embodiment, the batch period is carried out in a batch medium using 40-50 g of glycerol per liter, specifically 45 g of glycerol per liter as the basal carbon source, and the culture is carried out at 25. It is carried out at any temperature between 25 ° C. and 30 ° C. at ° C. for about 27-30 hours, or at 30 ° C. for about 23-36 hours, or for a culture time of 23-36 hours. Decreasing the concentration of glycerol in the batch medium will shorten the length of the batch period, whereas increasing the glycerol in the batch medium will result in a longer batch period. As an alternative to glycerol, glucose can also be used, for example, in approximately the same amount.

In a typical system of cell culture and POI expression, in which the fed-batch phase is followed by the fed-batch phase, specifically, the culture during the fed-batch phase is carried out for about 15 to 80 hours, about 15 to 70 hours, about. 15-60 hours, about 15-50 hours, about 15-45 hours, about 15-40 hours, about 15-35 hours, about 15-30 hours, about 15-35 hours, about 15-25 hours, or about 15 ~ 20 hours; preferably over any of about 20-40 hours. Specifically, the culture during the fed-batch period is carried out for about 80 hours, about 70 hours, about 60 hours, about 55 hours, about 50 hours, about 45 hours, about 40 hours, about 35 hours, about 33 hours, and about 30 hours. , Approximately 25 hours, approximately 20 hours, or approximately 15 hours.

Any such fed-batch culture of less than 120 hours, less than 100 hours, or less than 80 hours, which results in successful POI production, thereby resulting in a high yield, is referred to herein as " It is called "fast fermentation". Specifically, the volume-specific product formation rate (rP) is the amount of product (mg) (mg (L h) ^-1 ) formed per unit volume (L) and unit time (h). .. Volume-specific product formation rates are also referred to as space yield (STY) or volume productivity.

Specifically, fed-batch cultures are performed to achieve an air-time yield of ^{approximately 30 mg (L h) -1} (meaning 30 mg (L h) ^{-1 ± 5% or ± 10%).} Specifically, an air-time yield of about 30 mg (L h) ^-1 was achieved within a fed-batch of about 30 hours, specifically at least 27, 28, 29, 30, 31, 32, or 33 mg (L). h) ^-1 in less than 33 hours, 32 hours, 31 hours, 30 hours, 29 hours, 28 hours, 27 hours, 26 hours, or 25 hours. Can be achieved.

Specifically, the batch period is carried out as the first step a), and the fed-batch period is carried out as the second step b).

Specifically, in the second step b), a growth limiting amount of the supplementary carbon source is supplied, and the specific growth rate is set in ^{the range of 0.04h -1 to} 0.2h ^-1 , preferably 0. Feed medium kept below any of 2, 0.15 ^{, 0.1h -1} or 0.15h ^{-1 is used during the feeding period.}

Specifically, the method of batch culture and feeding culture is a yeast host cell, for example, yeast of either the genus Saccharomyces or the genus Pichia or the genus Komagataella, or Pichia. Yeasts from genus other than the genus, such as K. K. lactis, Z. Luke (Z. rouxii), P.M. P. stypitis, H. Polymorpha (H. polymorpha), or Y. Yeasts derived from Y. lipolytica, preferably methanol-utilizing yeast (Pichia pastoris) or Komagataela pastoris (Komagataella pastoris) are incorporated. Specifically, yeast is used in fast fermentation.

Specifically, the batch culture and fed-batch culture methods employ the pG1-x promoter, which is any of SEQ ID NOs: 37 to 44, preferably any of SEQ ID NOs: 45 to 76. In particular, the pG1-x promoter is characterized by SEQ ID NO: 39, preferably SEQ ID NO: 49.

Specifically, POIs are made at a transcription rate of at least 15% compared to the cell's native pGAP promoter.

According to specific embodiments, the basal carbon source differs from the supplemental carbon source, eg, quantitatively and / or qualitatively. Quantitative differences may result in different conditions for suppressing or relieving promoter activity.

According to a more specific embodiment, the basal carbon source and the supplemental carbon source preferably contain the same type of molecule or carbohydrate in different concentrations. According to a more specific embodiment, the carbon source is a mixture of two or more different carbon sources.

Any type of organic carbon suitable for use in eukaryotic cell cultures may be used. According to specific embodiments, the carbon source is a hexose, such as glucose, fructose, galactose or mannose, a disaccharide, such as saccharose, alcohol, such as glycerol or ethanol, or a mixture thereof.

Specifically, according to a preferred embodiment, the basal carbon source is selected from the group consisting of glucose, glycerol, ethanol, or mixtures thereof, and complex nutritional materials. According to a preferred embodiment, the basal carbon source is glycerol.

According to a more specific embodiment, the supplemental carbon source is a hexose, such as glucose, fructose, galactose and mannose, a disaccharide, such as saccharose, alcohol, such as glycerol or ethanol, or a mixture thereof. According to a preferred embodiment, the supplemental carbon source is glucose.

Specifically
a) The basal carbon source is selected from the group consisting of glucose, glycerol, ethanol, mixtures thereof, and complex nutritional materials;
b) The supplemental carbon source is a hexose, such as glucose, fructose, galactose or mannose, a disaccharide, such as saccharose, alcohol, such as glycerol or ethanol, or a mixture of any of the above.

The culturing step specifically involves culturing the cell line in the presence of the carbon source and thus in a culture medium containing the carbon source, or also in step b) of the supplemental carbon source. Includes culturing cell lines even in the absence.

The disinhibition (or induction) condition may be adequately achieved by specific means. The second step b) optionally employs a feed medium that does not supply a supplemental carbon source or supplies a limited amount of supplemental carbon source.

Specifically, the feed medium is a chemically defined medium that does not contain methanol.

The feed medium may be added to the culture medium in liquid form or in other alternative forms, such as solids, such as tablets or other means of continuous release, or in gas, such as carbon dioxide. However, according to a preferred embodiment, the limit of the supplemental carbon source added to the cell culture medium may still be zero. Preferably, under conditions of restricted carbon substrate, the concentration of the supplemental carbon source in the culture medium is 0 to 1 g / L, preferably less than 0.6 g / L, more preferably less than 0.3 g / L, more preferably. Is less than 0.1 g / L, preferably 1 to 50 mg / L, more preferably 1 to 10 mg / L, specifically preferably 1 mg / L, or less, eg, suitable standard. It falls below the detection limit determined as the residual concentration in the culture medium as measured in the assay, eg, when consumed by a growing cell culture.

In a preferred method, the residual amount in the cell culture provided by the limiting supplemental source is determined in the fermentation medium at the end of the production phase, or preferably in the output of the fermentation process at the time of harvesting the fermentation product. Below the detection limit.

Specifically, in the second step b), a growth limiting amount of the supplementary carbon source is supplied, and the specific growth rate is set to 0.001 h ^{-1 to} 0.2 h ^-1 , preferably 0.005 h ^-1. Incorporate feed medium, keeping within the range of ~ 0.15 h ^-1.

Sequence analysis of pG1 for TFBS associated with carbon source using MatInspector. pG1 (also referred to as P _Gth1) is first amplified and a length of -965～-1 position (965Bp, the sequence presented in Figure 6 (SEQ ID NO: 1, in particular, using SEQ ID NO: 9) ) Was cloned. The numbers point to TFBS and TFBS was selected for deletion (listed in Table 2). Related matrix families include F $ CSER (carbon source response element, shaded box), F $ ADR (yeast metabolism regulator, stippled box), F $ MGCM (monomer Gal4 class motif, black box) and F $ YMIG (yeast GC box protein, white box). Other TFBSs may also be affected by the deletion (detailed information on matrix matches is shown in Table 1). The black dashed box points to the major regulatory region of pG1 identified by screening for shortened pG1 mutants. The asterisks indicate the location of the prominent TAT (positions -390 to -374) motif, which is also selected for deletions and mutations. An alternative 5'side starting point for the shortened pG1 promoter variant is represented by an arrow and the length of the corresponding variant. Screening data for abbreviated pG1 promoter variants Under inhibitory and inducible growth conditions, pG1 (clone # 8, validated GCN is 1) or abbreviated pG1 variant (two clones each, three Geometric mean values of population specific eGFP fluorescence (fluorescence associated with cell volume) are shown for clones expressing eGFP under the control of (selected in sequel and pre-screening). Non-expressed wild-type methanol-utilizing yeast (P. pastoris) cells were used as a negative control. Samples were taken during suppressive preculture and after induction with feed beads for 24 and 48 hours. Screening data for TFBS-deficient and TFBS-TAT mutants Under inhibitory and inducible growth conditions, pG1 (clone # 8, verified GCN is 1) or pG1 mutant (nine or less) For clones expressing eGFP under the control of (pool-cultured clones in 3 wells), the geometric average of the population's specific eGFP fluorescence (fluorescence associated with cell volume) is shown. Wild-type methanol-utilizing yeast (P. pastoris) cells were used as a negative control. Screening data for pG1 duplicate variants Under suppressive and inducible growth conditions, pG1 (clone # 8, validated GCN is 1) or pG1 variant (9 or less clones in 3 wells) For clones expressing eGFP under the control of pool culture and prescreening), the geometric mean of the population's specific eGFP fluorescence (fluorescence associated with cell volume) is shown. Wild-type methanol-utilizing yeast (P. pastoris) cells were used as a negative control. Fed-batch culture of pG1 and pG1 variants expressing eGFP Relative eGFP fluorescence was measured from a bioreactor sample (diluted to similar biomass density) using a plate reader, batch culture (A) and fed-batch. It is shown over the feed time in culture (B) (the end point of each batch is 0). Clones expressing eGFP under the control of pG1 (# 8) were cultivated with pG1 deletion mutants (pG1-Δ2, SEQ ID NO: 211), TAT mutations (pG1-T16, SEQ ID NO: 257), and duplications (pG1-D1240). ) Compared to clones expressed under the control of mutant (SEQ ID NO: 49). pG1 and pG1-x promoter sequences Reference sequence sequence of pG1-x promoter

(Continued) Transcription factor sequence Rgt1 (PAS_chr1-3_0233) (SEQ ID NO: 261) Cat8-2 (PAS_chr4_0540) (SEQ ID NO: 262) Cat8-1 (PAS_chr1-3_0757) (SEQ ID NO: 263) Prior art is described in the sequence WO2013050551A1, pG1 (SEQ ID NO: 264), pG1a (SEQ ID NO: 265), pG1b (SEQ ID NO: 266), pG1c (SEQ ID NO: 267), pG1d (SEQ ID NO: 268), pG1e (SEQ ID NO: 269), or pG1f (SEQ ID NO: 270) SEQ ID NO: Using (A) standard fed-batch protocol, (B) fed-batch protocol with optimized space yield (“fast fermentation”), sourced from Maurer et al. (Microbial Cell Factories, 2006, 5:37). Fed-batch culture of 39 selected pG1-3 embodiments (pG1-D1240 (SEQ ID NO: 49)) in which an alternative scaffold protein is expressed as a model protein.

Detailed description of the invention

The special terms used throughout this specification have the following meanings:

As used herein, the term "carbon source", also also referred to as "carbon substrate", is a fermentable carbon substrate, typically a source of carbohydrates suitable as an energy source for microorganisms. For example, a source selected from the group consisting of alcohols containing monosaccharides, oligosaccharides, polysaccharides, glycerol, such as sources capable of metabolism by the host organism or production cell system, the source in purified form, the minimum. It shall mean a source in the medium, or a source supplied by a source, such as a polysaccharide material. The carbon source may be used as a single carbon source or as a mixture of different carbon sources according to the present invention.

A "basic carbon source", eg, a "basic carbon source" as used in accordance with the present invention, is typically a carbon source suitable for cell growth, eg, a nutrient for eukaryotic cells. The basal carbon source may be supplied by medium, eg, basal medium or composite medium, or may be supplied by a chemically defined medium containing a purified carbon source. The basal carbon source is typically an amount that results in cell proliferation, especially during the growth phase of the culture process, eg, at least 5 g of dry cell mass per liter, preferably at least 10 g of dry cell mass per liter, or 1 L. Cell dry mass per cell An amount that yields a cell density of at least 15 g, eg, an amount that exhibits greater than 90% viability, preferably greater than 95% viability in a standard subculture step. ..

According to the invention, the basal carbon source typically increases biomass, for example, when culturing a cell line at a high specific growth rate, for example, during the growth phase of the cell line during a batch or fed-batch culture process. Used in excess or surplus, understood as excess that results in energy to cause. This surplus is, in particular, a residual concentration in the fermentation broth that exceeds the limit of the supplemental carbon source (used under growth restriction conditions) and is measurable and typically of the limit. Achieve a residual concentration that is at least 10-fold, preferably at least 50-fold, or at least 100-fold when feeding the supplemental carbon source.

A "replenishment carbon source", eg, a "replenishment carbon source" as used in accordance with the present invention, typically facilitates the production of fermented products by the producing cell line, especially during the production phase of the culture process. It is a supplementary substrate. The production phase specifically follows the growth phase, for example in batch, fed-batch, and continuous culture processes. The supplemental carbon source may specifically be included in the feed of the fed-batch process. The supplemental carbon source is typically incorporated in cell cultures under carbon substrate limiting conditions, i.e., in cell cultures that use a limited amount of carbon source.

A "restricted amount" of carbon source or "restricted carbon source" is used herein to facilitate the production of fermented products by a producing cell line, especially in a culture process with a controlled growth rate below the maximum growth rate. It is understood to specifically refer to the type and amount of carbon substrate. The production phase specifically follows the growth phase, for example in batch, fed-batch, and continuous culture processes. The cell culture process may incorporate batch cultures, continuous cultures, and fed-batch cultures. Batch culture is a culture process in which a small amount of seed culture solution is added to a medium, and at the time of culturing, cells are proliferated without adding a further medium or draining the culture solution. Continuous culture is a culture process in which a medium is continuously added and discharged during culturing. Continuous culture also includes perfusion culture. Fed-batch culture, which is intermediate between batch culture and continuous culture, and is also called semi-batch culture, is in which a medium is continuously or sequentially added at the time of culturing, but unlike continuous culture, a culture solution is continuously added. It is a culture process that does not excrete.

Specifically, a fed-batch process based on feeding the growth-restricted nutrient substrate into the culture is preferred. Fed-batch strategies, including single-batch or repeated-batch fermentation, are typically used in bioindustry processes to reach high cell densities in the bioreactor. The controlled addition of carbon substrate directly affects the growth rate of the culture and helps avoid overflow metabolism or the formation of unwanted metabolic by-products. Under carbon source limiting conditions, the carbon source may specifically be included in the feed of the fed-batch process. As a result, the carbon substrate is supplied in a limited amount.

In the chemostat or continuous cultures described herein, the growth rate can also be tightly controlled.

Carbon source limits are understood herein specifically as the amount of carbon source required to keep a producing cell line under growth limiting conditions, eg, production phase or mode of production. Such limits are incorporated in the fed-batch process in which the carbon source is contained in the feed medium and fed to the culture at a small feed rate for sustained energy delivery, eg, biomass. POIs may be made while maintaining a small specific growth rate. The feed medium is typically added to the fermentation broth during the cell culture production phase.

The carbon source limit is determined, for example, by the residual amount of carbon source in the cell culture medium, which is below a predetermined threshold or below the detection limit measured in a standard (carbohydrate) assay. May be good. Residual amounts will typically be determined in the fermentation broth when recovering the fermentation product.

The carbon source limit is also the average feed rate of the carbon source to the fermenter per culture time, eg, the whole culture process, so as to determine the average amount calculated per hour. For example, it may be determined by defining an average feed rate, which is determined by the amount added over the culture period. This average feed rate is small to confirm the complete use of the supplemental carbon source by the cell culture, eg 0.6 g L ^{-1 h -1} (carbon supply per 1 L of initial fermentation capacity per hour per h). source g) ~25g L ^-1 h ^-1, preferably kept ^{1.6g L -1 h -1 ~20g L -1} h -1.

The limit of the carbon source may also be determined by measuring the specific growth rate, which is small during the production phase, eg, less than the maximum specific growth rate, eg, within a predetermined range. For example, keep within the range of ^{0.001h -1 to} 0.20h ^-1 , or 0.005h ^{-1 to} 0.20h ^-1 , preferably 0.01h ^{-1 to} 0.15h ^-1.

Specifically, use a feed medium that is chemically defined and does not contain methanol.

The term "chemically defined" for cell culture media, eg, minimal media or feed medium in the feeding process, is a culture medium suitable for cell culture of production cell lines in vitro, with chemical components and ( All of the poly) peptides shall mean known culture media. Typically, the chemically defined medium is completely free of animal-derived components and represents a pure and consistent cell culture environment.

As used herein, the term "cell line" refers to an established clone of a particular cell type that has acquired the ability to proliferate over time. The term "host cell line" is used to express a product of an endogenous or recombinant gene or metabolic pathway to produce a polypeptide or cell metabolite mediated by such a polypeptide. Point to. A "producing host cell line" or "producing cell line" is generally understood to be a cell line that is easy to use for culturing in a bioreactor to obtain the product of the production process, eg, POI. The term "eukaryotic host" or "eukaryotic cell lineage" shall mean any eukaryotic cell or organism that may be cultured to make a POI or host cell metabolite. It is well understood that the term does not include humans.

With respect to the host cell line, the term "cell culture" or "culture", also referred to as "fermentation", refers to the proliferation, differentiation, or proliferation of active or dormant cells in an artificial, eg, in vitro environment. It means the maintenance of cells in a controlled bioreactor, specifically according to methods known in the industry, under conditions suitable for sustaining viability.

When culturing a cell culture using the culture medium of the present invention, the cell culture is brought into contact with the medium or substrate in the culture tank under conditions suitable for supporting the culture of the cell culture. In certain embodiments, the culture medium described herein is used to culture cells according to standard cell culture methods well known in the art. Various aspects of the invention provide a culture medium that can be used to grow eukaryotic cells, specifically yeast or filamentous fungi.

The cell culture medium provides the nutrients needed to maintain the cells and grow them in a controlled, artificial in vitro environment. The characteristics and composition of the cell culture medium vary according to specific cell requirements. Important parameters include osmolality, pH, and nutrient formulation. The feeding of nutrients may be carried out continuously or discontinuously according to a method known in the art. The culture medium used according to the present invention is particularly useful for making recombinant proteins.

The batch process is a culture method in which all the nutrients required to culture the cells at the time of fermentation without further supply of nutrients are contained in the initial culture medium, whereas the batch period. In the subsequent fed-batch process, the feeding provides a feeding phase that feeds the culture with one or more nutrients. The purpose of nutrient feeding is to increase the amount of biomass and also the amount of recombinant protein. In most culture processes, the feeding method is essential and important, but the present invention, which utilizes the promoter of the present invention, is not restricted by any culture method.

In certain aspects, the method of the invention is a fed-batch process. Specifically, host cells transformed with a nucleic acid construct encoding the desired recombinant POI are cultured in growth medium and transferred to production medium to produce the desired recombinant POI.

In another embodiment, the host cells of the invention are cultured in a continuous manner, eg, by chemostat. The continuous fermentation process is characterized by a constant and continuous feeding rate of fresh culture medium to the bioreactor, with the same constant and continuous removal rate of the culture broth from the bioreactor. Remove at the same time. By keeping the culture medium, feeding rate, and removal rate at the same constant level, the culture parameters and conditions within the bioreactor remain constant.

Stable cell cultures described herein are those that maintain genetic properties, specifically high levels of POI production, eg, about 20 generations, preferably at least 30 generations, more preferably at least 40 generations. Most preferably, it is specifically understood to refer to a cell culture that remains at least at the μg level even after culturing for at least 50 generations. Specifically, it provides a stable recombinant host cell line that would be of great advantage when used for industrial scale fabrication.

The cell cultures of the present invention relate to, for example, feeding of nutrients, especially in terms of both volume and technique systems combined with a fed-batch or batch process, or a culture scheme based on a continuous or semi-continuous process (eg, chemostat). , Especially advantageous for methods on an industrial production scale.

The terms "expression" or "expression system" or "expression cassette" are used so that a host transformed or transfected with these sequences can produce the encoded protein or host cell metabolite. Refers to a nucleic acid molecule that contains the desired coding and control sequences within the operative linkage. The expression system may be integrated into the vector to carry out the transformation, or the involved DNA may be integrated into the host chromosome. Expression may refer to a secreted expression product, including a polypeptide or metabolite, or may refer to a non-secretory expression product.

The "expression construct" or "vector" or "plasmid" used herein is required for cloning recombinant nucleotide sequences, i.e. transcription of recombinant genes, and translation of their mRNAs in a suitable host organism. It is defined as a DNA sequence. Expression vectors or plasmids are usually starting points for autonomous replication in host cells, selectable markers (eg, genes that confer resistance to zeocin, kanamycin, G418, or hyglomycin), amino acid synthesis genes or antibiotics, etc. It contains a number of restriction enzyme cleavage sites, suitable promoter sequences, and transcription terminators, and these components are operably linked together. The terms "plasmid" and "vector" used herein include self-replicating nucleotide sequences as well as genomic integration nucleotide sequences.

The expression construct of the present invention specifically comprises the promoter of the present invention, which is operably linked to a nucleotide sequence encoding POI under transcriptional control of said promoter, which promoter encodes the POI. It does not naturally associate with the sequence.

The term "heterologous" as used herein with respect to a nucleotide or amino acid sequence or protein is foreign to a given host cell, i.e. "exogenous", eg, a compound not found in nature, or Refers to any of the compounds found naturally in a given host cell, eg, "endogenous", but in the context of a heterologous construct, eg, which recruits a heterologous nucleic acid. Heterologous nucleotide sequences found endogenously may also be made in an amount that is non-natural, eg, greater than what is expected intracellularly, or greater than what is naturally found intracellularly. Nucleic acids containing heterologous nucleotide sequences or heterologous nucleotide sequences probably encode the same protein that is sequenced differently from the endogenous nucleotide sequence but is found endogenously. Specifically, a heterologous nucleotide sequence is a nucleotide sequence that is not found in the same relationship as a host cell in nature. Any recombinant or artificial nucleotide sequence is understood to be heterologous. Examples of heterologous polynucleotides are nucleotide sequences that do not naturally associate with promoters according to the invention, eg, nucleotide sequences that yield hybrid promoters, or nucleotide sequences that are operably linked to coding sequences, as described herein. is there. As a result, hybrid or chimeric polynucleotides may be obtained. A further example of a heterologous compound is a polynucleotide encoding a POI in which an endogenous, naturally occurring POI-encoding sequence is normally not operably linked, a transcriptional control element, eg, an operably linked to a promoter of the invention. Is.

As used herein in the context of the present invention, the term "mutant" is intended to refer to any sequence with specific sequence identity or homology to an equivalent parent sequence. A variant specifically involves a change in size, eg, (terminal or non-terminal, eg, "interstitial", that is, a deletion or insertion within a nucleotide sequence, from the parent sequence. ) Any sequence derived via extension or fragmentation, mutation, hybridization (including sequence combinations).

The pG1-x promoter described herein is specifically an artificial variant of the native (wild-type) pG1 promoter. Some degree of sequence identity to the natural structure is observed, but the materials of the invention specifically refer to, for example, isolated nucleic acid sequences, amino acid sequences, expression constructs, transformed host cells, and recombinant proteins. , Methods, and uses are "artificial" or synthetic and are therefore not considered as a result of "laws of nature".

The promoter referred to herein as the "pG1-x promoter" is a variant of the pG1 promoter, the nucleotide sequence of which is made by mutagenesis of the pG1 promoter, which is used as the "parent" sequence for making the variant. can do. The pG1-x promoter comprises a promoter containing two, three, four or more copies of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

The series of pG1-x promoters may be, for example, any of the sequences exemplified in FIG. 6b, in particular the following sequence:
a) Any of SEQ ID NOs: 37-44, preferably SEQ ID NOs: 45-76;
b) Any of SEQ ID NOs: 77-80, preferably SEQ ID NOs: 81-112;
c) Any of SEQ ID NOs: 113-114, preferably SEQ ID NOs: 115-130;
d) Any of SEQ ID NOs: 131-132, preferably SEQ ID NOs: 133-148;
e) Any of SEQ ID NOs: 149-150, preferably SEQ ID NOs: 151-166;
f) Any of SEQ ID NOs: 167 to 168, preferably SEQ ID NOs: 169 to 184;
g) Any of SEQ ID NOs: 185 to 186, preferably SEQ ID NOs: 187 to 202;
h) Any of SEQ ID NOs: 203-204, preferably SEQ ID NOs: 205-220;
i) Any of SEQ ID NOs: 221 to 222, preferably SEQ ID NOs: 223 to 238;
j) SEQ ID NOs: 239-240, preferably any of SEQ ID NOs: 241-256; and k) SEQ ID NOs: 32-36 or SEQ ID NOs: 257-259.
Illustrated by a promoter comprising or consisting of any of these.

The pG1-x promoter is also a 3'fragment of any one of SEQ ID NOs: 37 to 202, deleting some or all of the major regulatory region from the 5'end to the first or 5'side. 3'fragment; preferably, the 5'end of any one of SEQ ID NOs: 37 to 202, 50, 100, 150, 200, 250, 300, 320, or 325 or less nucleotides are deleted. Also includes 3'fragments.

The pG1-x promoter is the same or increased promoter intensity and induction ratio as compared to the pG1 promoter.
-Promoter strength is increased at least 1.1-fold compared to the pG1 promoter and / or-Induction ratio is increased at least 1.1-fold compared to the pG1 promoter in the inducible state Promoter strength and induction It is characterized by having a ratio.

In particular, to maintain or improve the essential elements and functions of the promoter, for example, the illustrated pG1-x promoter, or size variant thereof, in particular an extension variant or fragment thereof, of FIG. 6b. Additional pG1-x variants can also be made using as the "parent" sequence for making mutants by mutagenesis of a particular region. The pG1-x promoter variant is derived from any of the exemplified pG1-x promoter sequences, for example by mutagenesis to create a sequence suitable for use as a promoter in a recombinant cell line. May be good. Such mutant promoters may be obtained from a library of mutant sequences by selecting library members with defined properties. Mutant promoters may have the same or improved properties, eg, increase the differential effect (particularly the induction ratio) under inhibitory and disinhibited conditions, to increase promoter strength, POI production. It may have properties with improved induction. The variant promoter is also a nucleotide sequence derived from a similar sequence, eg, a eukaryotic species other than methanol-utilizing yeast (Pichia pastoris), or a genus other than Pichia, eg, K. K. lactis, Z. Luke (Z. rouxii), P.M. P. stypitis, H. Nucleotide sequences derived from polymorpha (H. polymorpha) may also be included.

For example, the term "functionally active" as used herein with respect to a variant of the pG1-x promoter or a variant of the pG1-x promoter, or a variant of the pG1 promoter described herein. Mutation induction, specifically modification of the parent sequence by insertion, deletion, or substitution of one or more nucleotides within the sequence or at one or both of the distal ends of the sequence, which affects the activity of this sequence. It means a mutant sequence obtained as a result of a modification that does not affect (particularly, does not impair this). With respect to the pG1-x promoters described herein, function and activity are specifically characterized by the activity and intensity of the promoter, as well as the induction ratio.

The functionally active promoter variants described herein specifically exhibit promoter activity that is substantially the same (± 10%, or ± 5%) as, or greater than, the pG1 promoter. It is characterized by doing.

The functionally active promoter variants described herein are specifically by, for example, substantially the same induction ratio (± 10%, or ± 5%) as, or greater than, the pG1 promoter. It is characterized by exhibiting substantially the same regulatory properties as measured.

As used herein, the term "promoter" refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. Promoter activity may be evaluated by its transcription efficiency. This may be determined directly by measuring the amount of mRNA transcribed by the promoter, for example via Northern blotting, or indirectly by measuring the amount of gene product expressed by the promoter. May be.

The pG1-x promoters described herein specifically induce, regulate, or otherwise mediate or regulate the expression of coding DNA. The promoter DNA and the coding DNA may be derived from the same gene, different genes, the same organism, or different organisms.

The pG1-x promoter described herein is specifically understood as a regulatory promoter, particularly a promoter regulatory by a carbon source, with different promoter intensities in the inhibitory and inducible states. Will be done.

The intensity of the promoter of the present invention relates specifically to its transcriptional intensity, which is represented by the efficiency of transcriptional induction that occurs frequently or infrequently in the promoter. The higher the transcription intensity, the greater the frequency of transcription that occurs in the promoter. Promoter strength, which determines how often a given mRNA sequence is transcribed, effectively gives some genes a greater transcriptional priority over others. It is important because it results in a large transcript concentration. Genes encoding large amounts of protein are typically, for example, relatively strong promoters. Since only one transcription task can be performed on RNA polymerase at a time, it must be prioritized to make the task efficient. Differences in promoter strength are selected to allow this priority.

According to the present invention, the regulated promoter is relatively potent in a fully induced state, which is typically understood as a state of near maximal activity.

Relative intensity is generally determined for each pGAP promoter of an equivalent promoter, such as the pG1 promoter, or a standard promoter, such as a cell used as a host cell. Transcription frequency is generally understood as a transcription rate determined by the amount of transcript in a suitable assay, eg RT-PCR or Northern blotting. For example, the transcriptional strength of a promoter according to the invention is determined within a host cell of methanol-utilizing yeast (P. pastoris) and compared to the native pGAP promoter of methanol-utilizing yeast (P. pastoris).

The intensity of a promoter expressing a gene of interest is generally understood as the ability to support expression intensity or high expression levels / rates. For example, the expression and / or transcriptional intensity of the promoter of the invention is determined within a host cell of methanol-utilizing yeast (P. pastoris) and compared to the native pGAP promoter of methanol-utilizing yeast (P. pastoris). ..

Comparative transcriptional strength using the pGAP promoter as a reference material (reference material) refers to standard means, eg, measuring the number of transcripts, eg, microarrays, or other means in cell culture. That, for example, may be determined by measuring the quantity of each gene expression product in the recombinant cell. Illustrative tests are illustrated in the Examples section.

In particular, the transcription rate may be determined by the transcription intensity on the microarray or by quantitative real-time PCR (qRT-PCR). In this case, the condition of high growth rate is determined by the microarray or qRT-PCR data. Differences in expression levels between conditions with low growth rates or with different media compositions, and large signal intensities compared to the native pGAP promoter are shown.

The expression rate may be determined, for example, by the expression level of a reporter gene, for example, eGFP.

The pG1-x promoters described herein are reflected in a host cell by at least 15% transcription rate or transcription intensity as compared to a native pGAP promoter, sometimes referred to as the "homologous pGAP promoter". Provides a large transfer strength. Preferably, the transcription rate or intensity is at least 20%, specifically preferably at least 20%, as compared to, for example, the native pGAP promoter determined within eukaryotic cells selected as the host cell for making POI. At least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, and at least 100%, and / or greater, for example, at least 150% or at least 200%. is there.

The native pGAP promoter typically induces the expression of the gap gene, which encodes glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a constitutive promoter present in most organisms. GAPDH (EC 1 \ 2 \ 1 \ 12), a key enzyme in glycolysis and gluconeogenesis, plays a vital role in carbohydrate metabolism by catabolism and assimilation.

Specifically, the native pGAP promoter is used in recombinant eukaryotic cells in the same manner as in native eukaryotic cells of the same species or strain, including unmodified (non-recombinant) or recombinant eukaryotic cells. It is active. Such native pGAP promoters are generally understood to be endogenous promoters and therefore allogeneic to eukaryotic cells and are used as standard or reference promoters for comparison purposes.

For example, the native pGAP promoter of methanol-utilizing yeast (P. pastoris) has, for example, the sequence shown in FIG. 13: the native pGAP promoter sequence of methanol-utilizing yeast (P. pastoris) (GS115) (SEQ ID NO: 260). , An unmodified endogenous promoter sequence in methanol-utilizing yeast (P. pastoris) used to control the expression of GAPDH in methanol-utilizing yeast (P. pastoris). When methanol-utilizing yeast (P. pastoris) is used as a host for producing POI according to the present invention, the transcriptional strength or rate of the promoter according to the present invention can be adjusted to that of such methanol-utilizing yeast (P. pastoris). Compare with the native pGAP promoter.

As another example, the native pGAP promoter of S. cerevisiae is used in S. cerevisiae to control GAPDH expression in S. cerevisiae, as it is used in S. cerevisiae. It is an unmodified endogenous promoter sequence. When S. cerevisiae is used as a host for producing POI according to the present invention, the transcriptional strength or rate of the promoter according to the present invention is combined with the natural pGAP promoter of such budding yeast (S. cerevisiae). Compare.

Therefore, the relative expression or transcriptional intensity of a promoter according to the invention is typically compared to the native pGAP promoter of cells of the same species or strain used as a host for making POI.

The term "regulated" as used herein with respect to the pG1-x or pG1 promoter refers to eukaryotic cells during the growth phase of a fed-batch culture in the presence of an excess of carbon source (nutrient or basal substrate). During the production phase of the production cell line, which is suppressed within, eg, when the carbon content is reduced, eg, when feeding a growth-restricted carbon source (nutrient or supplementary substrate) into the culture according to a fed-batch strategy. , Refers to a promoter that is desuppressed to result in strong promoter activity. In this regard, the term "regulated" refers to the desuppression of the promoter by the consumption, reduction, lack or depletion of carbon, or the addition of a carbon source restricted to be more easily consumed by cells. It is understood as "adjustable by carbon source restriction" or "adjustable by glucose restriction".

The functionally active pG1-x promoter described herein is a relatively potent, regulatory promoter that is silenced or suppressed under cell growth conditions (proliferation phase) and under production conditions (proliferation phase). During the production phase), it is a promoter suitable for inducing POI production in the producing cell line by being activated or desuppressed and therefore by a restricted carbon source.

Specifically, the promoters described herein are regulated by a carbon source and are compared with differential promoter intensities determined in tests comparing their intensities in the presence of glucose and in the presence of glucose limitation. It also shows that a large glucose concentration, preferably at least 10 g per liter, preferably at least 20 g per liter, is also suppressed. Specifically, promoters according to the invention are fully induced at restricted glucose and glucose threshold concentrations that fully induce the promoter, the threshold being less than 20 g per liter, preferably less than 10 g per liter, 1 g per liter. Less than, more than less than 0.1 g per liter, or less than 50 mg per liter, preferably the complete transcription intensity is at least 50% of the native homologous pGAP promoter, for example, at a glucose concentration of less than 40 mg per liter.

Preferably, the induction ratio is understood as a differential promoter intensity determined by the initiation of POI production when switching to induction conditions below a defined carbon source threshold and compared to the intensity in the suppressed state. Will be done. Transfer strength is generally understood as strength in a fully induced state, that is, strength exhibiting activity close to maximum under disinhibition release conditions. The differential promoter strength is determined, for example, according to the efficiency or yield of POI production in the recombinant host cell line under reinhibition conditions compared to the suppression conditions, or otherwise by the amount of transcript. .. Regulated promoters according to the invention are preferably at least 2-fold, more preferably at least 5-fold, even more preferably at least 10-fold, more preferably at least, in the desuppressed state as compared to the suppressed state. It has a differential promoter strength of 20-fold, more preferably at least 30, 40, 50, or 100-fold, and is also understood as an induction multiple.

The term "sequence identity" of a variant compared to its parent sequence means that two or more nucleotide sequences are, to some extent, up to nearly 100% identical or conservative base pairs at the corresponding positions. Indicates the degree of identity (or homology) in the sense of having. Homologous sequences typically have at least about 50% nucleotide sequence identity, preferably at least about 60% identity, more preferably at least about 70% identity, more preferably at least about 80%. , More preferably at least about 90% identity, more preferably at least about 95% identity.

For example, a "percentage of identity (%)" with respect to a nucleotide sequence of a promoter or gene is sequenced and, if necessary, a gap is introduced to achieve the maximum percentage of sequence identity, with any conservative substitution. It is defined as the percentage of nucleotides in a candidate DNA sequence that are identical to the nucleotides in the DNA sequence, after not being considered part of the sequence identity. Alignment aimed at determining the percent nucleotide sequence identity can be achieved in a variety of aspects within the art, eg, using commercially available computer software. One of ordinary skill in the art can determine suitable parameters for measuring the alignment, including any algorithm required to achieve maximum alignment over the overall length of the sequence being compared. For the purposes of the present invention, blastn is set to the following exemplary parameters: Word Size: 11; Expect value: 10; Gap cost: Existence = 5, Extension = 2; Filter = low complexity activated; Match / Missatch-Score: 3; Filter String: L; m, NCBI BLAST program version 2.2.29 (January 6, 2014) is used to determine sequence identity between two nucleotide sequences.

The term "mutagenesis" as used in the context of the present invention results in mutants of the nucleotide sequence, eg, by insertion, deletion, and / or substitution of one or more nucleotides, and is non-coding or coding. It shall refer to a method of obtaining the variant with at least one change in the region. Mutagenesis may be via random, semi-random, or site-directed mutations. A specific pG1-x promoter variant is derived from the pG1 promoter sequence by a mutagenesis method using the pG1 nucleotide sequence as the parent sequence. Such mutagenesis methods include methods of manipulating nucleic acids or synthesizing nucleotide sequences in de novo using sequence information of the pG1 promoter as a template. Specific mutagenesis methods apply rational promoter manipulation.

The pG1-x promoter may be made by mutagenesis of the pG1 promoter and is a variant of the pG1-x promoter described herein with the help of standard techniques and is a functionally active variant. Variants, including the body, may also be made. The promoter may be modified, for example, to produce promoter variants with altered expression levels and regulatory properties. For example, a promoter library may be prepared by mutagenesis of selected promoter sequences, which can be used as the parent molecule to analyze variants for their expression, eg, under different fermentation strategies, and be appropriate. Gene expression in eukaryotic cells may be fine-tuned by selecting a suitable mutant. A synthetic library of variants may be used, for example, to select promoters that meet the requirements for making selected POIs. Such mutants can increase expression efficiency in eukaryotic host cells and exhibit differential expression under carbon source-rich and limiting conditions. Typically, a large randomized gene library is created that has a large gene diversity and can be specifically selected according to the desired genotype or phenotype.

Some of the preferred pG1-x promoters described herein are size variants of the pG1 promoter, containing more than one copy of a particular element or region of the promoter, or one of the pG1 promoters. Contains one or more (same or different) fragments.

Specific mutagenesis methods are point mutations of one or more nucleotides in a sequence, particularly tandem point mutations, eg, at least two, three, four in the nucleotide sequence of a promoter. It results in point mutations that alter one, five, six, seven, eight, nine, ten, and even more contiguous nucleotides. Such mutations are typically deletions, insertions, and / or substitutions of at least one of one or more nucleotides. The promoter sequence may be mutated at the distal end, especially within the 5'side region, but this is less than 50% of the nucleotide sequence and is highly advanced as long as the promoter activity is not substantially lost. It may be variable. The promoter sequence can be specifically mutated within the major regulatory region, but has greater sequence identity to the parent major regulatory region of pG1, in particular greater sequence identity to the parent core regulatory region, eg, at least 80%. Is preferable. Within the major regulatory region, but outside the core regulatory region, sequence variability may be large enough to obtain less than 80% sequence identity.

The core regulatory region specifically incorporates SEQ ID NO: 2 and SEQ ID NO: 3, and an intermediate region between SEQ ID NO: 2 and SEQ ID NO: 3, which represents a transcription factor binding site (TFBS).

The nucleotide sequence identified as SEQ ID NO: 2 comprises at least a portion of the TFBS recognized by Rgt1, Cat8-1, and Cat8-2.

The nucleotide sequence identified as SEQ ID NO: 3 comprises at least a portion of the TFBS recognized by Rgt1, Cat8-1, and Cat8-2.

Specifically, the nucleotide sequence between SEQ ID NO: 2 and SEQ ID NO: 3 (intermediate sequence) may be mutated to a non-homologous sequence (eg, sequence identity less than 50%). Alternatively, it may be deleted.

Any mutations in SEQ ID NO: 2 and SEQ ID NO: 3 are specifically conservative, i.e., for example, to maintain (or improve) recognition by their respective transcription factors. When manipulating such conservative mutants, the sequence identity within the nucleotide sequence of SEQ ID NO: 2 and / or SEQ ID NO: 3 is at least 90%, preferably at least 95%.

The major regulatory region comprises or consists of the nucleotide sequence identified by SEQ ID NO: 5. Such regions include a core regulatory region and an additional non-core regulatory region that contains the essential elements of the pG1 promoter and may be mutated to some extent to make the pG1-x promoter described herein. ..

Specific regions for site-directed mutagenesis are, for example, the non-core regulatory regions of the pG1 or pG1-x promoter (inside or outside the major regulatory region). However, specific mutants may also be prepared by mutagenesis methods that direct the core regulatory region of the promoter, as long as some sequence identity is maintained, so as to maintain promoter function. More specific areas are outside or inside the main regulatory area. Specifically, the promoter is, for example, a core regulatory region and one or more regions of the pG1 promoter, or an alternative (natural or artificial) promoter, eg, any promoter other than the pG1 promoter, which is a translation of the pG1 promoter. A hybrid comprising a translation initiation site in the 3'side region of the promoter, specifically the 3'end containing at least 10 nucleotides at the end or at least 15 nucleotides at the end, which is a promoter that can be used to replace the start site. It may contain the nucleotide sequence of the body.

A specific mutation refers to the duplication of a selected region (or motif) of the pG1 promoter, eg, a T-motif or an extended T-motif. Such selected motifs may be extended or shortened by additional nucleotides at one or both distal ends of the motif, or within the motif. The native pG1 sequence comprises a TAT motif (SEQ ID NO: 14) consisting of T15 following nucleotide "T" followed by "A". Such a TAT motif, 5'-TATTTTTTTTTTTTTTTT-3'(SEQ ID NO: 22), has been found to have a positive effect on promoter strength, which is still possible by duplicating the TAT motif. At least of the TAT motif, using an extended T motif (eg, TAT motif), including the same TAT motif, or an alternative T motif, at least the T13 motif (SEQ ID NO: 12), which may be augmented. It may still be augmented by inserting two, three, or four copies.

The present invention also includes nucleotide sequences that hybridize to the pG1-x promoter under strict conditions.

As used in the present invention, the term "hybridization" or "hybridization" means that two nucleic acid sequences are annealed to each other under appropriate conditions with stable and specific hydrogen bonds. It is intended to mean the process of forming the main chain. Hybridization between two complementary or fully complementary sequences depends on the operating conditions used, and in particular on the rigor. Strictness may be understood to delineate the degree of homology, and the higher the rigor, the greater the percentage of homology between sequences. Rigidity may be defined in particular by the base composition of the two nucleic acid sequences and / or by the degree of mismatch between these two nucleic acid sequences. By varying conditions, eg, salt concentration and temperature, a given nucleic acid sequence may hybridize only with its exact complement (highly rigorous) or with any related sequence. May (low degree of rigor). Increasing temperature or decreasing salt concentration can tend to increase the selectivity of the hybridization reaction.

The phrase "hybridizing under strict hybridization conditions" as used in the present invention is understood to preferably refer to hybridizing under certain rigorous conditions. In a preferred embodiment, "strict hybridization conditions" are conditions in which the homology of the two nucleic acid sequences is at least 70%, preferably at least 80%, preferably at least 90%, i.e., in this hybridization. Conditions under which hybridization is possible only if the resulting double strand contains at least 70%, preferably at least 80%, preferably at least 90% AT and CG bonds. Is.

Rigidity may depend on reaction parameters, such as the concentration and type of ionic molecular species present in the hybridization solution, the nature and concentration of the denaturant, and / or the hybridization temperature. Appropriate conditions can be determined by those skilled in the art, for example, as described in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, 1989).

The term "isolated" or "isolated" as used herein with respect to nucleic acids, POIs, or other compounds is sufficient from a naturally associated environment to be present in "substantially pure" form. It shall refer to the compound separated into. "Isolated" means the exclusion of the presence of artificial or synthetic mixtures with other compounds or materials, or impurities that do not interfere with the underlying activity, eg, impurities that may be present due to incomplete purification. Does not necessarily mean. In particular, the isolated nucleic acid molecule of the present invention also means that it also comprises a chemically synthesized nucleic acid molecule, in particular a nucleic acid molecule that does not naturally occur in methanol-utilizing yeast (P. pastoris) or any other organism. It also means that a nucleic acid molecule referred to here as "artificial" is included. With respect to the nucleic acids of the invention, the term "isolated nucleic acid" or "isolated nucleic acid sequence" is optionally used. When applied to DNA, the term refers to a DNA molecule that has been isolated from a directly adjacent sequence within the naturally occurring genome of the organism from which it is derived. For example, an "isolated nucleic acid" may include a DNA molecule inserted into a vector, eg, a plasmid or viral vector, or a DNA molecule integrated into the genomic DNA of a prokaryotic or eukaryotic cell or host organism. An "isolated nucleic acid" (either DNA or RNA) may further represent a molecule that is made directly by biological or synthetic means and separated from other components present at the time of its preparation.

As used herein, the term "operably linked" means that the function of one or more nucleotide sequences of a nucleotide sequence on a single nucleic acid molecule, eg, a vector, is present on said nucleic acid molecule. Refers to an association in a manner that is influenced by at least one other nucleotide sequence. For example, a promoter is operably linked to the coding sequence of a recombinant gene if it is possible to carry out expression of that coding sequence. As a further example, a nucleic acid encoding a signal peptide can act on a nucleic acid sequence encoding a POI if it is capable of expressing a protein, eg, a precursor or mature protein of a mature protein, in a secretory form. It is connected. Specifically, such nucleic acids operably linked to each other may be linked directly, i.e., between the nucleic acid encoding the signal peptide and the nucleic acid sequence encoding the POI, without any additional element or nucleic acid sequence. Good.

When a promoter controls transcription of a coding sequence, it is typically understood that the promoter sequence is operably linked to the coding sequence. If the promoter sequence does not naturally associate with the coding sequence, its transcription is not regulated by the promoter in native (wild-type) cells, or the sequence has been recombined with different contiguous sequences.

As used herein, the term "protein of interest (POI)" refers to a polypeptide or protein produced by recombinant techniques in a host cell.
More specifically, the protein may be a polypeptide that is not naturally occurring in the host cell, i.e. a heterologous protein, or a polypeptide that is natural to the host cell, i.e. a protein that is homologous to the host cell. Good, but for example, by transformation with a self-replicating vector containing a POI-encoding nucleic acid sequence, or by recombination of one or more copies of the POI-encoding nucleic acid sequence into the genome of the host cell at the time of integration. , Or by recombination modification of one or more regulatory sequences that control the expression of the gene encoding POI, such as the promoter sequence. In some cases, the term POI as used herein also relates to the product of any metabolism by a host cell mediated by a recombinantly expressed protein.

The POI may be specifically recovered from a cell culture in purified form, eg, substantially pure.

The terms "substantially pure" or "purified" as used herein are at least 50% (w / w), preferably at least 60%, 70%, 80%, 90%, or 95%. A compound of, for example, a nucleic acid molecule or a preparation containing a POI. Purity is measured by a method suitable for the compound (eg, chromatography, polyacrylamide gel electrophoresis, HPLC analysis, etc.).

As used herein, the term "recombination" shall mean "prepared by genetic manipulation or a result thereof". Therefore, a "recombinant microorganism" includes at least one "recombinant nucleic acid". Recombinant microorganisms specifically include an expression vector or cloning vector, or are genetically engineered to contain a recombinant nucleic acid sequence. A "recombinant protein" is prepared by expressing each recombinant nucleic acid in a host. A "recombinant promoter" is a genetically engineered non-coding nucleotide sequence described herein that is suitable for its use as a functionally active promoter.

In general, the recombinant nucleic acids or organisms referred to herein may be made by recombinant methods well known to those of skill in the art. According to the present invention, conventional molecular biology, microbiology, and recombinant DNA techniques within the art may be incorporated. Such techniques are well described in the literature. See, for example, Maniatis, Fritsch, and Sambrook, "Molecular Cloning: A Laboratory Manual," Cold Spring Harbor (1982).

According to a preferred embodiment of the invention, a recombinant construct is obtained by ligating the promoter and the gene involved into a vector or expression construct. These genes can be stably integrated into the host cell genome by transforming the host cell with such a vector or expression construct.

Expression vectors may include, but are not limited to, cloning vectors, modified cloning vectors, and specifically designed plasmids. The preferred expression vector used in the present invention may be any expression vector suitable for expression of the recombinant gene in the host cell, and is selected according to the host organism. The recombinant expression vector may be any vector that can be replicated or integrated into the genome of the host organism, also called the host vector.

Suitable expression vectors typically contain additional regulatory sequences suitable for expressing the DNA encoding the POI in the eukaryotic host cell. Examples of regulatory sequences include operators, enhancers, ribosome-binding sites, and sequences that control the initiation and termination of transcription and translation. The regulatory sequence may be operably linked to the DNA sequence for expression.

To allow expression of the recombinant nucleotide sequence in the host cell, the expression vector puts a promoter according to the invention adjacent to the 5'end of the coding sequence, eg, secreting the gene of interest (GOI) or POI. May be brought upstream of the signal peptide gene that enables. Transcription is thus regulated and initiated by this promoter sequence.

As used herein, the term "signal peptide" is used specifically to refer to a natural signal peptide, a heterologous signal peptide, or a hybrid of a natural signal peptide and a heterologous signal peptide, and specifically a host producing a POI. It may be different from the organism or may be the same species. The function of the signal peptide is to allow POI to be secreted into the endoplasmic reticulum. The signal peptide is usually a short (3-60 amino acid long) peptide chain that directs the transport of the protein out of the cell membrane, thereby facilitating the separation and purification of heterologous proteins. Some signal peptides are cleaved from the protein by signal peptidase after the protein has been transported.

Exemplary signal peptides include a signal sequence derived from S. cerevisiae alpha-zygote factor prepropeptide, as well as the acid phosphatase gene (PHO1) and extracellular protein X (EPX1) of Saccharomyces cerevisiae (P. pastoris). ) Is a signal peptide (Heiss et al., 2015; WO20140679926A1).

For example, to demonstrate the function of the sequence of involvement, an expression vector containing one or more of the regulatory elements (eg, the pG1-x promoter and optionally the signal sequence) is constructed to express POI. It may be driven and the expression yield is compared to constructs with conventional regulatory elements. To generate a wide variety of different POIs at high levels, the identified nucleotide sequences are amplified by PCR using specific nucleotide primers and cloned into expression vectors, eg, yeast vectors and methanol-transformed yeast (P). A strain of yeast) may be used to transform into a eukaryotic cell line. To estimate the effect of the pG1-x promoters described herein on the amount of recombinant POI thus produced, eukaryotic cell lines were introduced into each cell with the conventional pG1 or pGAP promoter. May be cultured by shaking flask experiment and fed-batch or promoter fermentation as compared to the strain containing. In particular, promoter selection has a significant effect on the production of recombinant proteins.

POIs are made using recombinant host cell lines by culturing transformants, thereby isolating the expressed product or biotransform from the culture and optionally purifying it by a suitable method. Can be obtained in a suitable medium.

Transformants according to the present invention can be obtained by introducing such vector DNA, for example, plasmid DNA, into a host and selecting a transformant that expresses POI or a host cell metabolite in high yield. .. By the methods conventionally used to process host cells and transform eukaryotic cells, such as the electric pulse method, protoplast method, lithium acetate method, and modifications thereof, they incorporate foreign DNA. Is possible. Methanol-utilizing yeast (P. pastoris) is preferably transformed by electroporation. Preferred transformation methods for the uptake of recombinant DNA fragments by microorganisms include transformation by chemical transformation, electroporation, or protoplastization. Transformants according to the present invention are obtained by introducing such vector DNA, for example, plasmid DNA, into a host and selecting a transformant that expresses the protein or host cell metabolite involved in high yield. be able to.

A number of different methods for making POIs that follow the methods of the invention are preferred. The substance is a culture of transformed cells in which eukaryotic host cells are transformed with an expression vector carrying a protein involved and recombinant DNA encoding at least one of the regulatory elements described above. May be expressed, processed and optionally secreted by preparing, growing the culture, inducing transcription and production of POI, and recovering the product of the fermentation process.

Host cells according to the invention are preferably examined for their expressiveness or yield by the following tests: ELISA, activity assay, HPLC, or other suitable test.

The present invention specifically allows for pilot or industrial scale fermentation processes. For industrial process scales, a capacity of at least 10 L, specifically at least 50 L, preferably at least 1 m ³ , preferably at least 10 m ³ , and most preferably at least 100 m ³ will be used.

^{For example, about 0.02 to 0.15 h -1 in fed-batch culture with a reactor capacity of 100 L to 10 m 3} or more, or fermenter capacity of about 50 to 1000 L or more, with the aid of typical process times of several days. Industrial scale production conditions, which refer to a continuous process with a dilution rate of.

Suitable culture methods may include in-bioreactor cultures starting with a batch phase, followed by a short exponential fed-batch period at a large specific growth rate, followed by a fed-batch period at a small specific growth rate. Good. Another suitable culture method may include a continuous culture phase at a small dilution rate following the batch period.

A preferred embodiment comprises fed-batch culture for high yield POI production following batch cultures that result in biomass.

The host cell lines described herein are cultured in a bioreactor under proliferation conditions to a dry weight of at least 1 g of cells per liter, more preferably at least 10 g of dry weight of cells per liter, preferably at least per liter. It is preferable to obtain a cell density of 20 g of dry cell weight. It is advantageous to bring such yields of biomass production on a pilot or industrial scale.

Growth media that allow the accumulation of biomass, specifically basal growth media, typically include a carbon source, a nitrogen source, a source for sulfur, and a source for phosphoric acid. Typically, such media further contains trace elements and vitamins and may further contain amino acids, peptones, or yeast extracts.

Preferred nitrogen sources include NH ₄ H ₂ PO ₄ , or NH ₃ or (NH 4) ₂ SO ₄ .

Preferred sulfur sources include sulfonyl ₄ , or (NH4) ₂ SO ₄ or K ₂ SO ₄ .

Preferred phosphate sources include NH ₄ H ₂ PO ₄ , or H ₃ PO ₄ or _{Na H 2} PO ₄ , KH ₂ PO ₄ , Na ₂ HPO ₄ or K ₂ HPO ₄ .

Further typical medium components include KCl, CaCl ₂ , and trace elements such as Fe, Co, Cu, Ni, Zn, Mo, Mn, I, B.

Preferably, the medium is supplemented with _{vitamin B 7.}

A typical growth medium for methanol-utilizing yeast (P. pastoris) contains glycerol, sorbitol, or glucose, NH ₄ H ₂ PO ₄ , sulfonyl ₄ , KCl, CaCl ₂ , biotin, and trace elements.

During the production phase, the production medium is specifically used with only a limiting amount of supplemental carbon source.

Preferably, the host cell line is cultured in an inorganic medium with a suitable carbon source, which further significantly simplifies the isolation process. Examples of preferred inorganic media are available carbon sources (eg glucose, glycerol, sorbitol, or methanol), salts containing large elements (potassium, magnesium, calcium, ammonium, chlorides, sulfuric acid, phosphoric acid). And salts containing trace elements (copper, iodide, manganese, molybdic acid, cobalt, zinc, and iron salts, and boric acid) and optionally, for example, vitamins or amino acids that complement nutritional requirements. It is an inorganic medium.

Specifically, the cell or cell depends on the nature of the expression system and the protein being expressed, eg, whether the protein is fused to a signal peptide, and whether the protein is soluble or membrane-bound. Incubate under conditions suitable to carry out the expression of the desired POI that can be purified from the culture medium. As will be appreciated by those skilled in the art, culture conditions will vary depending on the type of host cell and factors including the particular expression vector used.

A typical production medium contains a supplemental carbon source, as well as additional NH ₄ H ₂ PO ₄ , sulfonyl ₄ , KCl, CaCl ₂ , biotin, and trace elements.

For example, the feed of supplemental carbon sources added to the fermented product may include carbon sources with up to 50% by weight of available sugar. A low feed rate of the replacement medium will limit the inhibitory effect of the product or by-product on cell proliferation, thus allowing high product yields based on substrate supply.

Fermentation is preferably carried out at a pH in the range of 3 to 7.5.

A typical fermentation time is about 24 to 120 hours, with the temperature in the range of 20 ° C to 35 ° C, preferably 22 to 30 ° C.

POI is preferably expressed with the aid of conditions that result in yields of at least 1 mg / L, preferably at least 10 mg / L, preferably at least 100 mg / L, and most preferably at least 1 g / L.

It is understood that the methods disclosed herein may further comprise culturing the recombinant host cells, preferably under conditions that allow expression of POI in secretory form or as an intracellular product. Will be done. The POI or host cell metabolite produced by recombination can then be isolated from cell culture medium and further purified by techniques well known to those of skill in the art.

POIs made according to the present invention can typically be isolated and purified using modern techniques, including increasing the concentration of the desired POI and / or decreasing the concentration of at least one impurity.

If the POI is secreted from the cells, the POI can be isolated and purified from the culture medium using modern techniques. Secretion of the recombinant expression product from the host cell is generally because the product is recovered from the culture supernatant rather than from the complex mixture of proteins resulting from the destruction of yeast cells and the release of intracellular proteins. It is advantageous for reasons including facilitating the purification process.

To obtain a cell extract containing the desired POI, from which the POI is isolated and purified, the cultured transformed cells may also be ultrasonically disrupted. It may be broken mechanically, enzymatically, or chemically.

As isolation and purification methods for obtaining recombinant polypeptide or protein products, methods such as utilizing soluble differences, such as salting out and solvent precipitation, methods utilizing molecular weight differences, eg, limiting. Filtering and gel electrophoresis, methods that utilize charge differences, such as ion exchange chromatography, methods that utilize specific affinity, such as affinity chromatography, methods that utilize hydrophobic differences, such as reversed-phase high performance liquids. Chromatography and methods that take advantage of isoelectric focusing, such as isoelectric focusing, may be used.

The highly purified product is essentially free of contaminating proteins and preferably has a purity of at least 90%, more preferably at least 95%, and / or at least 98%, up to 100%. The purified product may be obtained by purifying the cell culture supernatant, or may be obtained from a cell disruption product.

Isolation and purification methods include the following standard methods: cell disruption (when POI is obtained intracellularly), precision filtration or tangential flow filtration (TFF), or separation and washing of cells (crushed material) by centrifugation. Purification of POI by precipitation or heat treatment, activation of POI by enzymatic digestion, purification of POI by chromatography, eg ion exchange (IEX), hydrophobic interaction chromatography (HIC), affinity chromatography, size exclusion Precipitation of POI by (SEC) or HPLC chromatography, concentration and washing via an ultrafiltration process is preferred.

The isolated and purified POI can be identified by conventional methods such as Western blot, HPLC, activity assay, or ELISA.

The POI can be any eukaryote, prokaryote, or synthetic polypeptide. The POI may be a secretory protein or an intracellular protein. The invention also provides recombinant production of functional homologues, functional equivalent variants, derivatives, and biologically active fragments of naturally occurring proteins. The functional homologue preferably has, or corresponds to, the functional features of the sequence.

The POIs referred to herein are preferably homologous or heterologous products to eukaryotic host cells for therapeutic, prophylactic, diagnostic, analytical, or industrial use. You may.

The POI is preferably a heterologous recombinant polypeptide or protein produced in eukaryotic cells, preferably yeast cells, preferably as a secreted protein. Examples of preferably made proteins are immunoglobulins, immunoglobulin fragments, aprotinins, tissue factor pathway inhibitors, or other protease inhibitors, and insulin or insulin precursors, insulin analogs, growth hormones, interleukins, tissue plus. Minogen activator, transformative growth factor a or b, glucagon, glucagon-like peptide 1 (GLP-1), glucagon-like peptide 2 (GLP-2), GRPP, factor VII, factor VIII, factor XIII, platelet-derived growth Factor 1, serum albumin, enzymes, such as lipases or proteases, or functional homologues, functional equivalent variants, derivatives, and biologically active fragments that have similar functions to native proteins. The POI may be structurally similar to the natural protein, with the addition of one or more amino acids to one or both or side chains of the C and N ends of the natural protein, one or more in the natural amino acid sequence. Substitution of one or more amino acids at different sites, deletion of one or more amino acids at one or both ends of a natural protein, or at one or more sites within an amino acid sequence, or one within a natural amino acid sequence It may be derived from a natural protein by inserting one or more amino acids at the above sites. Such modifications are well known for some of the proteins mentioned above.

The POI can also be selected from substrates, enzymes, inhibitors, or cofactors that result in a biochemical reaction within the host cell, which yields the product of the biochemical reaction or cascade of multiple reactions. That is, for example, the purpose is to obtain metabolites of host cells. Exemplary products can be vitamins such as riboflavin, organic acids, and alcohols, which can be obtained in increased yield after expression of a recombinant protein or POI according to the invention.

In general, the host cell expressing the recombinant product can be any eukaryotic cell suitable for recombinant POI expression.

Examples of preferred mammalian cells include BHK, CHO (CHO-DG44, CHO-DUXB11, CHO-DUKX, CHO-K1, CHOK1SV, CHO-S), HeLa, HEK293, MDCK, NIH3T3, NS0, PER. C6, SP2 / 0, and VERO cells.

Examples of preferred yeast cells used as host cells according to the present invention are Saccharomyces (eg, Saccharomyces cerevisiae), Pichia (eg, P. pastoris). , Or P. methanolica), Komagataella (K. pastoris), K. pseudopastoris, or K. phaffii, Hansenula. -Hansenula polymorpha, Yarrowia lipolytica, Saccharomyces stypitis (Schefferomyces stipitis), or Kluyberomyces lactis (Schefferomyces stipitis), or Kluyberomyces lactis.

In recent literature, methanol-utilizing yeast (Pichia pastoris) has been divided into Komagataella pastoris, Komagataella pastoris, and Komagataella pseudopastoris, and Komagataela pseudopastoris. Here, methanol-utilizing yeast (Pichia pastoris) is used for Komagataela pastoris, Komagataela paffii, and Komagataela pseudopastoris for Komagataela pastori.

Preferred yeast host cells are methylotrope yeast, such as Pichia or Komagataella, such as methanol-utilizing yeast (Pichia pastoris), or Komagataela pastoris, or K. K. phaffii, or K. Derived from K. pseudopastoris. Examples of hosts include yeast, such as methanol-utilizing yeast (P. pastoris). Examples of methanol-utilizing yeast (P. pastoris) strains are CBS 704 (= NRRL Y-1603 = DSMZ 70382), CBS 2612 (= NRRL Y-7556), CBS 7435 (= NRRL Y-11430), CBS 9173 to 9189 (CBS strain: CBS-KNAW Fundamental Biodiversity Center, Centraal bureau voor Schemacultures, Utrecht, The Netherlands), and DSMZ 70877 (German).
Collection of Microorganisms and Cell Cultures), but also includes strains from Invitrogen, such as X-33, GS115, KM71, and SMD1168. Examples of S. cerevisiae strains include W303, CEN. Includes PK and BY series (EUROSCARF collection). All of the strains listed above have been used to create transformants and successfully express heterologous genes.

A preferred yeast host cell according to the present invention, for example, a host cell produced by P. pastoris or S. cerevisiae, is a yeast that is different from the production host, such as P. pastoris or Saccharomyces cerevisiae. It contains a heterologous or recombinant promoter sequence that may be derived from the S. cerevisiae strain. In another specific embodiment, a host cell according to the invention comprises a recombinant expression construct according to the invention, comprising a promoter derived from the same genus, species, or strain as the host cell.

According to the present invention, it is possible to provide a host cell line by methanol-utilizing yeast (P. pastoris) containing the pG1-x promoter sequence described herein operably linked to a nucleotide sequence encoding POI. preferable.

When the POI is a protein of the same species as the host cell, i.e., a naturally occurring protein in the host cell, expression of the POI in the host cell is modulated by exchanging its native promoter sequence with a promoter sequence according to the invention. You may.

This objective is achieved, for example, by transforming a host cell with a recombinant DNA molecule that contains a homologous sequence of target genes that allows site-specific recombination, a promoter sequence, and a selectable marker suitable for the host cell. You may. Site-specific recombination shall be performed to operably link the promoter sequence to the nucleotide sequence encoding the POI. This results in the expression of POI by the promoter sequence according to the invention instead of the native promoter sequence.

Specifically, the pG1-x promoter preferably increases the promoter activity in the light of the POI's native promoter sequence.

According to a specific embodiment, in the POI production method, a recombinant nucleotide sequence encoding POI, which is a plasmid suitable for integration into the genome of a host cell, is provided in a single copy or multiple copies per cell. Incorporate recombinant nucleotide sequences. The recombinant nucleotide sequence encoding the POI is also provided by a self-replicating plasmid in a single copy or multiple copies per cell.

The preferred method described herein employs a plasmid that is a eukaryotic expression vector, preferably a yeast expression vector. Expression vectors may include, but are not limited to, cloning vectors, modified cloning vectors, and specifically designed plasmids. The preferred expression vector used in the present invention may be any expression vector suitable for expression of the recombinant gene in the host cell, and is selected according to the host organism. The recombinant expression vector may be any vector, also called a host vector, that can replicate or integrate within the genome of the host organism, eg, a yeast vector carrying a DNA construct according to the invention. .. Preferred yeast expression vectors are for expression in yeast selected from the group consisting of methylotrophic yeasts represented by the genus Hansenula, Pichia, Candida, and Torulopsis. It is an expression vector.

In the present invention, it is preferable to use a plasmid derived from pPICZ, pGAPZ, pPIC9, pPICZ alpha, pGAPZ alpha, pPIC9K, pGAPHis, or pPUZZLE as a vector.

According to a preferred embodiment of the invention, the gene involved is ligated into a vector to give a recombinant construct. By transforming a host cell using such a vector, these genes can be stably integrated into the host cell genome. The gene-encoded polypeptide is made using a recombinant host cell line by culturing the transformant, thereby making the expressed POI isolated from the culture and suitable for the expressed product. Purification can be obtained by method, in particular by the method of separating POI from contaminating proteins, in a suitable medium.

The expression vector may contain one or more phenotypic selectable markers, such as genes encoding proteins that confer antibiotic resistance or impose autotrophic requirements. Yeast vectors are generally origins of replication derived from yeast plasmids, self-replicating sequences (ARS), or sequences used for integration into the host genome, promoter regions, sequences for polyadenylation, for transcription termination. Contains the sequence of, and a selection marker.

Ligate each of the DNA sequence and regulatory elements, such as the pG1-x promoter, and the gene, promoter, and terminator encoding the POI, and insert them into the appropriate vector containing the information required for integration or host replication. The procedure used to do so is well known to those of skill in the art, eg, J. Mol. Described by Sambrook et al. (A Laboratory Manual, Cold Spring Harbor, 1989).

Vectors that use regulatory elements and / or POIs according to the invention as integration targets first prepare a DNA construct containing the entire regulatory element and / or POI-encoding DNA sequence, and then this fragment is suitable. It is understood that it may be constructed by inserting it into an expression vector, or it may be constructed by sequentially inserting a DNA fragment containing genetic information about an individual element and then ligating it. Let's do it.

A multicloning vector that has a multicloning site and can incorporate a desired heterologous gene into the multicloning site to result in an expression vector can also be used in accordance with the present invention. Within the expression vector, the promoter is located upstream of the POI gene and regulates gene expression. In the case of a multicloning vector, the promoter is located upstream of the multicloning site in order to introduce the POI gene into the multicloning site.

The DNA constructs prepared to obtain recombinant host cells according to the invention may be prepared synthetically via established standard methods, such as the phosphoramidite method. DNA constructs also prepare, for example, genomes or cDNA libraries and hybridize using synthetic oligonucleotide probes according to standard techniques (Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, 1989). This may be a genome-derived DNA construct or a cDNA-derived DNA construct obtained by screening for a DNA sequence encoding all or part of the polypeptide of the present invention. Finally, the DNA construct is optionally a fragment derived from synthesis, a fragment derived from the genome, or a fragment derived from cDNA, which corresponds to various parts of the entire DNA construct according to standard techniques. It may be a DNA construct that is a mixture of a synthetic DNA construct and a genome-derived DNA construct prepared by annealing, or it may be a DNA construct that is a mixture of a synthetic-derived DNA construct and a cDNA-derived DNA construct. It may be a DNA construct that is a mixture of a DNA construct derived from a genome and a DNA construct derived from cDNA.

In another preferred embodiment, the yeast expression vector can be stably integrated into the yeast genome, for example by homologous recombination.

Transformed host cells according to the invention, obtained by transforming cells with a regulatory element and / or POI gene according to the invention, are preferably first cultured under conditions that allow a large number of cells to grow efficiently. May be good. When preparing a cell line to express POI, the culture method is chosen to produce the expression product.

The above description will be more fully understood by reference to the examples below. However, such examples merely represent a method of carrying out one or more aspects of the invention and should not be read as limiting the scope of the invention.

[Example]
Example 1
Shortening of the 5'side of pG1 reveals the major regulatory region of pG1 The natural (wild type) pG1 promoter is the methanol-utilizing yeast (P. pastoris) (Komagataella phaffii) CBS2612 strain (CBS). Strains: CBS-KNAW Fundamental Biodiversity Center, Centraal bureau voor Schimmelscultures, Utrecht, The Netherlands). As determined by Sanger sequencing and subsequent BLAST analysis, the pG1 promoter sequence of CBS2612 is GS115 (Invitrogen) (upstream of PAS_chr1-3_0011) and CBS7435 (upstream of P7435_Chr1-0007), or K.K. It had greater than 95% sequence identity to each region within the genomic sequence of the K. pastoris DSMZ70382 strain (DSMZ strain: German Collection of Microorganisms and Cell Cultures) (upstream of PIPA00372). At the time of analysis of the genomic region of pG1, the GTH1 gene was annotated with respect to the starting point in the CBS7435 (P7435_Chr1-0007) strain and the DSMZ70382 (PIPA00372) strain differently from those in the GS115 (PAS_chr1-3_0011) strain. Do you get it. In contrast to GS115 and CBS2612, in the genomic sequences of the other two strains, the coding sequence is annotated to begin 36 bp further downstream.

Starting from the alternative 5'side positions, -858, -663, -492, -371, -328, -283, -211, and -66, to identify the regulatory region of pG1 involvement. , To position -1, eight abbreviated pG1 variants were cloned from CBS2612 (see FIG. 1; numbering is based on locus PAS_chr1-3_0011, which is the starting point of the GTH1 gene). Suppressive properties (glycerol feed beads) of these shortened promoter variants screened for eGFP expression in deep well plates and compared to the original 965 bp form of pG1, as described in Example 8. And the inducing properties (glucose feed beads) were investigated (Fig. 2). Under the suppression conditions, no difference in eGFP signal was found for mutants of all lengths, indicating that promoter suppression was not restricted in any of the shortened mutants. After 48 hours of induction, expression capacity maintained full functionality for promoter variants up to 328 bp in length. The 283 bp mutant remained about two-thirds as strong as the original pG1 promoter. The two shortest length mutants (211 and 66bp) were considered to be nearly non-functional. These results suggest that the region between positions -400 and -200 contains important regulatory features.

Example 2
High-density TFBS, which is expected to be associated with a carbon source, matrix the pG1 promoter sequence (1000 bp upstream of gene PAS_chr1-3_0011) using a Genomatix MatInspector that marks the major regulatory region of the pG1 promoter. We searched for matrix families belonging to the "fungi" group and the "general core promoter element" group. We found 111 estimated TFBSs belonging to 46 different matrix families (Table 1). The most common matrix families in the sequence analyzed are the monomeric Gal4 class motif (F $ MGCM, 12 binding sites), homeodomain-containing transcriptional regulators (F $ HOMD, 6 binding sites). , The fungal basic leucine zipper family (F $ BZIP, 5 binding sites), and the yeast GC box protein (F $ YMIG, 5 binding sites). Extremely dense TFBS binding sites were detected between positions -400 and -200, and about two-thirds (18 of 28) of the mentioned TFBS (the most common matrix family) were these. Was in the position of. For general core promoter elements, neither yeast-related motifs nor fungal-related motifs were identified by MatInspector, but the TATA box can be found at position -26.

For example, it is a prominent motif at positions -390 to -375 and is referred to as TAT14 because of its sequence 5'-TATTTTTTTTTTTTTTTT-3'(SEQ ID NO: 21), or its sequence 5'-TATTTTTTTTTTTTTTTT-3. A motif called TAT15 was identified for'(SEQ ID NO: 22). It is known that such poly (A: T) tracts within the promoter region have a negative effect on nucleosome binding and stimulate TF binding at nearby sites within yeast.

Example 3
Transcription factors associated with carbon sources, Mxr1, Rgt1, Cat8-1, Cat8-2, and Mig1, have been shown to be important for the regulatory properties of pG1 glucose-dependent or carbon-source-dependent. Predicted transcription factor binding sites were selected for further analysis (see Figure 1 and Table 2). Overlap-extension PCR was used to generate pG1 mutants in which each region was deleted. Table 3 lists all selected TFBSs and points to all TFBSs (detailed list in Table 2) that are affected by the deletion (partially). For some deletions (eg, Δ9 and Δ10), some nucleotides in each TFBS were not deleted in order to keep nearby TFBSs functional and to consider their effects separately. left.

All TFBS-deficient and TAT mutants were screened for eGFP expression under inhibitory (glycerol feed beads) and inducible conditions (glucose feed beads) as described in Example 8 (Figure). 3). It is important to consider that individual TF / TFBS are usually not sufficient to fulfill promoter regulation. Deletion of TFBS also implies that promoter sequences can be affected by conjugation of newly formed sequences, changes in distance between TFBSs, or changes in higher-order properties (chromatin organization). .. Also, due to other adjacent TFBSs, the same TFBS at different positions in the promoter may perform different functions. At nearby TFBSs, TFs may act synergistically and may limit the binding of other TFs due to steric hindrance.

Four different carbon source-related TF families: yeast metabolism regulator (F $ ADR; matrix: F $ ADR1.01), monomeric Gal4 class motif (F $ MGCM; matrix: F $ RGT1.01,) F $ RGT1.02), carbon source responsive element (F $ CSRE, matrix: F $ CSR.01, F $ SIP4.01), and yeast GC box protein (F $ YMIG; matrix: F $ MIG1.01). And F $ MIG1.02) were deleted within the pG1 promoter mutant (see Tables 2 and 3). The corresponding transcription factors in S. cerevisiae are Dr1, Rgt1, Ship4 / Cat8, and Mig1, respectively.

Carbon source-dependent promoters are regulated by glucose suppression and / or induction via carbon sources other than carbohydrates or other sugars. Glucose suppression is mainly achieved by the protein kinase complex Snf1, the transcriptional repressor Mig1, and the protein phosphatase 1. In Saccharomyces cerevisiae, downstream factors regulate, for example, respiratory genes (Hap4), gluconeogenic genes (Cat8, Ship4), and glucose transporters (Rgt1).

Methanol-utilizing yeast (P. pastoris) has two Mig1 homologues, called Mig1-1 and Mig1-2, the latter of which probably acts as an inhibitor of carbon catabolism. When glucose is available, Mig1 acts as an inhibitor, whereas Rgt1 acts as a transcriptional activator. To perform its inhibitory function, Mig1 is dephosphorylated and translocated into the nucleus, where it recruits co-inhibitors Ssn6 and Tap1.

Under restricted glucose, Rgt1 is dephosphorylated and acts as a transcriptional repressor. In S. cerevisiae, the function of Rgt1 is regulated by its phosphorylation state (Rgt1 has four phosphorylation sites), and the regulation of promoter induction is typically seen for transcriptional repressors. As it is, it does not require dissociation of Rgt1.

In S. cerevisiae, the carbon source-responsive zinc finger transcription factor, Dr1, is required for transcriptional activation of the glucose-suppressing alcohol dehydrogenase (ADH2) gene. Homologs of Adr1 in Pichia pastoris (P. pastoris) is a key regulators of methanol metabolism are Mxr1 (PAS_chr4_0487), to methanol, essential for the induction of strong P _AOX, positive active It was reported to be a metabolic factor. The TFBS core motif reported for Mxr1, 5'CYCC3', is consistent with any F $ ADR 1.01 site found within the pG1 promoter sequence.

In S. cerevisiae, transcriptional activators Ship4 and Cat8 bind to the carbon source response element (CSER) and function to induce the expression of gluconeogenic genes. In methanol-utilizing yeast (P. pastoris), two homologues of ScCat8: Cat8-1 (PAS_chr2-1_0757) and Cat8-2 (PAS_chr4_0540) can be found, both of which are also best for ScSip4. It is a blastp hit. Although Cat8-2 has little similarity to ScCat8, it plays a potentially important role under disinhibition conditions.

Example 4
Deletion variants of the pG1 promoter reveal TFBS that contributes to its suppression and induction Five previously identified deletion variants located upstream (5'side) of the major regulatory region of pG1 Of (see the dashed box in FIGS. 1 and 2), the variants pG1-Δ1, pG1-Δ2, and pG1-Δ4 are thought to have a beneficial effect on promoter strength, but are missing. The demutants pG1-Δ3 and pG1-Δ5 had no effect on the expression of GFP as compared to the original pG1 promoter (SEQ ID NO: 9). This result suggests that shortening of the promoter on the 5'side may be beneficial in manipulating pG1. Deletions of TFBS within the major regulatory region of pG1 (pG1-Δ6 to pG1-Δ12; see FIG. 1 and Table 2) had different effects on eGFP expression, but both had inhibitory properties. No increase in induction was shown without loss. Therefore, it is hypothesized that the major regulatory region of pG1 needs to be maintained within the engineered pG1 promoter mutant to retain its tight regulation. Therefore, in Example 1, much lower induction was observed under restricted glucose without this region (pG1-328 and pG1-283; FIG. 2).

Mig1 binding sites were deleted at pG1-Δ3, pG1-Δ4, pG1-Δ10, and pG1-Δ11 (F $ MIG1.02 was deleted at Δ3 and F at Δ4, Δ10, and Δ11). $ MIG1.01 was deleted), but here pG1-Δ10 and pG1-Δ11 also include deletions of F $ ADR1.01 and F $ RGT1.02, respectively. For Δ3, a slight enhancement of inhibition was found, while for Δ4, no change in inhibition was observed, but eGFP levels were enhanced after induction.

The relaxation of inhibition seen for Δ10 and the reduction of promoter induction by Δ10 and Δ11 are also F $ RGT1 binding sites within this region (F $ RGT1.01 and F $ RGT1.02 deleted at Δ9 and Δ11). ) May be related. Mig1 may also have played a bifunctional role in the regulation of pG1: two MIG1 genes have been found in methanol-utilizing yeast (P. pastoris) (MIG1-1, MIG1- 2) It was shown that it is regulated in the opposite direction to the availability of glucose.

In the mutant pG1-Δ1, deletion of F $ ADR1.01 increased eGFP levels, but Mxr1 (a positive regulator of methanol metabolism in Pp and a homologue of ScADR1) binding site. Deletion of, on the contrary, would be expected to weaken the promoter. The combination of the deletion of F $ ADR1.01 with the deletion of F $ MIG1.01 at pG1-Δ10 alleviated the inhibition of the promoter by glycerol and weakened its induction, which was the lack of Mig1 TFBS. The final response to the loss.

In the variants pG1-Δ6 (two sites), pG1-Δ7, pG1-Δ8, pG1-Δ11, and pG1-Δ12, the binding site F $ RGT1.02 within the major regulatory region was deleted, and in Δ9, F $ RGT1.01 was deleted. Mutants harboring a deletion of the paired F $ RGT site 1.02 (Δ6; binding site on the reverse chain is accompanied by a 7 bp shift) showed slight relaxation of repression and reduced induction. Mutants Δ7 and Δ8 contain very close F $ RGT 1.02 sites, where the former F $ RGT 1.02 site is on the minus chain and the latter F $ RGT 1.02 site is plus. Although on the chain, Δ8 also contains a deletion of the F $ SIP4.01 site. The former (Δ7) showed a slight relaxation of inhibition and an increase in induction, whereas the induction by the latter (Δ8) was much weaker (but did not alter promoter inhibition). This points to a strong role of the transcriptional activators Cat8-1 and / or Cat8-2 (the most potent homologue of ScSip4) in the induction of pG1. Mutant Δ9 is placed in close proximity to F $ RGT1.01 and F $ CSER. It was created to delete the TFBS (binding site on the reverse chain) of 01, but a significant loss of inhibition was due to the binding of Rgt1, Cat8-1, and / or Cat8-2 to these TFBSs. It indicates that there is a powerful role to tightly control pG1 through. Deletion of F $ RGT1.02 in mutant pG1-Δ12 did not affect eGFP expression performance. Interestingly, transcription of CAT8-2 was strongly upregulated under restricted glucose compared to glucose residue, but under test conditions, transcriptional regulation of RGT1 and CAT8-2 was not made.

Example 5
pG1 promoter strength depends on the poly (A: T) tract TAT14 The TAT motif is located approximately 80 bp upstream (5', eg, positions -390 to -374) of the major regulatory region of pG1. Repeated sequencing of the 5'side region of GTH1 in CBS2612, CBS7435, or GS115 of methanol-utilizing yeast (P. pastoris) resulted in the detection of 15 ± 1 T in the TAT motif. To elucidate its effect on promoter performance, TAT14 motifs were selected for deletions (pG1-ΔTAT14) and mutations (T16, T18, and T20; to pG1-T16, pG1-T18, pG1-T20). Primers (see Primers # 37-42 in Table 4) were initially designed to obtain T18, T20, and T22, but mutants of different lengths (T16, T20, and T18, respectively). Obtained and used. Deletion of the TAT14 motif resulted in a decrease in GFP signal, whereas its elongation increased the expression intensity of pG1. This indicates that the use of the elongated TAT14 motif is beneficial for the manipulation of pG1.

Example 6
Partial sequence duplication of the major regulatory region of pG1 significantly improves its expression intensity by PCR amplification of two sequence fragments (-472-188 and -472--1), as well as restriction site PstI and Two duplicate variants of the pG1 promoter (pG1-D1240 (SEQ ID NO: 49) and pG1-D1427 (SEQ ID NO: 85); numbers are the respective promoters, by insertion using BglII (positions 509-514 and 525-530). (Representing the length of the mutant) was created. The overlapping interval begins upstream of the pG1-Δ5 deleted TFBS in the first variant (pG1-D1240) and ends behind the major regulatory region of pG1 while the second overlapping sequence (pG1-D1427). ) Reach the 3'end of the pG1 promoter. These mutants were screened for eGFP expression in the same manner as described for TFBS-deficient and TAT14 mutants (see Example 8). Both overlapping mutants showed enhanced inhibition under excess glycerol and enhanced induction under restricted glucose (Fig. 4).

The stability of the duplicate mutant clone, pG1-D1240 # 3, after transformation was investigated by performing three consecutive batch cultures, which is equivalent to about 20 generations, without selective pressure. Expression of eGFP was stable over the entire culture time (data not shown). In comparison, a typical bioreactor process with methanol-utilizing yeast (P. pastoris) _{begins and flows at OD 600} = 1 (about 0.2-0.4 g YDM per liter) in batches. After the addition, it ends with about 100 g of YDM per liter, which spans about 10 generations.

Example 7
Verification of performance of pG1 promoter mutants in fed-batch bioreactor culture In order to verify the performance of promoter mutants produced under bioprocess conditions, some mutants were modified to change the expression performance of their eGFP. Based on, selected for fed-batch culture: pG1-Δ2 (SEQ ID NO: 211) is the most enhanced variant upstream of the major regulatory region, pG1-T16 (SEQ ID NO: 257) and pG1-D1240. (SEQ ID NO: 49) showed high eGFP expression levels under restricted glucose without losing promoter inhibition under glycerol conditions. Bioreactor cultures (Prielhofer et al., 2013), starting with the glycerol batch period and followed by fed-batch with optimized air yield, were performed on each clone and control pG1 strain # for expression of eGFP. Compared with 8 (see Figure 5 and Table 5).

Fed-batch fermentation was carried out in a DASGIP reactor with a final working capacity of 0.7 L.

The following media were used.

PTM ₁ trace element salt stock solution per liter,
CuSO ₄ · 5H ₂ O of 6.0 g, NaI of 0.08g, MnSO ₄ · H ₂ O of 3.36 g, Na of _{0.2g 2 MoO 4 · 2H 2 O} , 0.02g of H ₃ BO _3, CoCl ₂ of 0.82 g, ZnCl ₂ in 20.0 g, FeSO of 65.0g ₄ · 7H ₂ O, biotin 0.2 g, and 5.0ml of _{H 2 SO 4 (95% ~98} %)
Contained.

Glycerol batch medium per liter
2g of citric acid monohydrate _{(C 6 H 8 O 7 ·} H 2 O), glycerol 39.2g, NH ₄ H ₂ PO ₄ of _{12.6g, MgSO 4 · 7H 2 O} , 0 to 0.5g .9g of KCl, CaCl ₂ · 2H ₂ O of 0.022 g, containing PTM1 trace elements salts stock solution of 0.4mg of biotin, and 4.6 ml. HCl was added to bring the pH to 5.

Glucose fed medium is per liter
Glucose monohydrate 464g, MgSO ₄ · 7H ² O of 5.2 g, 8.4 g of KCl, CaCl ₂ · 2H ₂ O in 0.28 g, PTM1 trace elements biotin 0.34 mg, and 10.1mL It contained a salt stock solution.

Dissolved oxygen was controlled to DO = 20% at a stirrer speed of 400-1200 rpm.
The aeration rate was 24 L h ^-1 for air, the temperature was controlled at 25 ° C., and the pH setting point 5 was _{controlled by adding NH 4} OH (25%).

To initiate fermentation, 400 mL batch medium was sterilized and filtered into a fermenter and the starting optical concentration (OD600) was set to 1 from the selective preculture of each methanol-utilizing yeast (P. pastoris) clone. Inoculated. After a batch period of about 25 hours (reaching a dry biomass concentration of about 20 g / L), start with an exponential feed over 7 hours and apply glucose limiting infusion for 13 hours at a constant feed rate of 15 g / L. The final dry biomass concentration of about 100 g / L was obtained. Samples were collected during batch and fed-batch periods and analyzed for eGFP expression using a plate reader (Infinite 200, Tecan, CH). Therefore, the sample was diluted to an optical concentration (OD600) of 5. The results are shown in FIG. 5 as relative fluorescence (FL / r) per bioreactor.

Real-time PCR was used to analyze the gene copy numbers of these three clones, resulting in a GCN of 1 for all of them (data not shown). All pG1 mutants exhibited good inhibition during batching and strong expression in the induced state (Table 5). Under bioreactor conditions, a strong improvement in the duplicate mutant pG1-D1240 could be verified, with clone pG1-D1240 # 3 at the end of feeding at 50% of GFP fluorescence compared to pG1. It showed an increase. At the end of the batch, the signal was already increased, but the induction ratio was slightly higher than the original pG1. Apart from screening, clone pG1-Δ2 # 3 slightly increased the signal at the end of the batch, but weakened the signal by about 10% at the end of feeding. The TAT14 mutant clone, pG1-T16 # 3, showed the strongest signal at the end of the batch, but was inferior to the duplicate mutant at the end of the feed and improved by about 20% over control pG1 # 8. Was reached, similar to the screening results. The different inducing behavior of the clones during the batch phase is explained by desuppression due to the reduction of glycerol concentration throughout the batch phase (see Figure 5A). Overall, fed-batch culture was able to confirm most of the results obtained by small-scale screening.

Achievements and Conclusions Gene promoters with carbon source-dependent regulation are suitable for bioprocess applications because their production phase is isolated from proliferation. Improvements in potential promoter-based protein production can also be achieved by finding optimal growth conditions (eg, growth rate, feeding strategy) and directly manipulating promoter sequences (eg, mutations, deletions). It can also be reached by.

Several pG1 promoter variants were constructed with reduced length, with TFBS deletions, TAT motif mutations, and fragment duplication. This identified the major regulatory region of pG1 containing its important TFBS. An analysis of the deletion of the TFBS, which lacked two motifs consisting of F $ RGT1 and F $ CRE, which bind at the same position on the reverse strand, was performed with the transcription factors Rgt1 and Cat8-1 and / or. It indicates that Cat8-2 plays an essential role in the suppression and induction of pG1. Deletion of the first part (pG1-Δ8: -293 to -285 positions; RGT1: (+)-310-299, CSER: (-)-299 to -285) weakens promoter induction, while Deletion of the second part (pG1-Δ9: -275-261; RGT1: (-)-275-259, CSER: (+)-276-260) reduced promoter repression. .. This identified a regulatory motif that is essential and characteristic for the regulation of pG1.

Under other conditions, such as excess glucose or methanol-induced conditions, the roles of the transcriptional regulators Mig1 (F $ MIG1) and Mxr1 (F $ ADR1) may be more important. Other transcription factors that bind within or near this region may also contribute to the regulation of pG1.

Poly (A: T) tracts, known to play a role in the promoter sequence, are TAT motifs within pG1 and are located upstream of the major regulator region (eg, positions -390 to -375). It was possible to show that the TAT motif produced is essential for the intensity of pG1. Extension of this motif to T16, T18, and T20 had a positive effect on promoter performance.

Deletion variants of pG1 revealed that shortening on the 5'side could be equally beneficial for promoter manipulation. Deletion of TFBS of Mxr1, Mig1, Rgt1, and Cat8 upstream of the major regulatory region of pG1 improved eGFP expression, but this effect was seen for 5'shortened promoter variants. There wasn't.

The two mutants with partial sequence duplication reached a significant enhancement of expression capacity compared to wild-type pG1.

Significantly different features of good expression performance of pG1, which is a solid basis for valid promoter manipulation: 5'side shortening, use of TAT motif, and assignment of arbitrary mutation / extension, and fragment duplication I was able to. The performance of the pG1 mutant in small-scale screening could be successfully verified in fed-batch culture.

Abbreviation:
CRE: Carbon source response element, F $: Fungus-specific TF matrix, GCN: Gene copy count, GOI: Target gene, Pp: Methanol-utilizing yeast (Pichia pastoris), Sc: Saccharomyces cerevisiae, TF : Transcription factor, TFBS: Transcription factor binding site, YDM: Yeast dry mass Example 8
Inhibition, induction, pG1-x expression level (expression level compared to pG1), determination of induction ratio Promoter intensity and induction ratio compared to the pG1 promoter can be determined by the following standard assay: Methanol The P. pastoris strain is screened in a deep 24-well plate at 25 ° C. with 2 mL of culture per well, shaking at 280 rpm. Glucose feed beads (6 mm, Kuhner, CH) are used to create glucose-restricted growth conditions. Flow cytometry is used to analyze cells for eGFP expression during inhibition (YP + 1% glycerol, exponential growth phase) and induction (YP + 1 feed beads over 20-28 hours). The specific eGFP fluorescence is calculated from the fluorescence intensity and forward scattering for at least 3000 data points of the flow cytometric data. Backscatter is a relative measure of cell volume. The specific GFP fluorescence is the fluorescence intensity (FI) divided by the forward scattering (FSC) to the 1.5th power, that is, ^{equal to FI / FSC 1.5} (Hohenblom, H., N. Barth, and D. Matanovich (2003), Assessing vivivity and cell-associated product of recombinant protein producing, Pichia pastoris withJo2, flow cytometry. From this data, the geometric mean of population specific fluorescence is used and standardized by reducing the background signal of wild-type non-producing methanol-utilizing yeast (P. pastoris) cells. The specific eGFP fluorescence under glycerol conditions is referred to as "inhibition" and the specific eGFP fluorescence under restricted glucose conditions (glucose feed beads) is referred to as "induction". Therefore, only suppression and induction values of the same screening as well as flow cytometry measurements can be compared and used for calculations. To determine the relative pG1-x promoter intensity, the eGFP of the pG1-x promoter in the induced state by dividing the induced value of the strain containing the pG1-x promoter by the induced value of the strain containing the original pG1 promoter. Expression levels were compared to the original pG1 promoter. The induction ratio is calculated by dividing the induction value of the same strain / promoter by the suppression value. Inhibition, induction, relative pG1-x promoter intensities, and induction ratios for some promoter variants are shown in Table 6.

A further example is that by using the pG1-x promoter comprising or consisting of the nucleotide sequence of SEQ ID NO: 49, the model protein (POI) is transformed into the unmodified pG1 promoter in P. pastoris. Criteria; It is demonstrated that it was produced with a much higher yield (increase factor greater than 3.5 times; feeding experiment) compared to SEQ ID NO: 7).

Example 9
Comparison of "Fast Fermentation" with Standard Fermentation Summary: pG1-3 aspects of SEQ ID NO: 39 by using a fed-batch protocol with optimized air-batch yield instead of using a standard fed-batch regimen. Under the control of the promoter by (pG1-D1240 (SEQ ID NO: 49)), a significant reduction in fermentation time could be obtained for the expression of an alternative scaffold protein as a model protein.

Under the control of pG1-D1240 (SEQ ID NO: 49), clones expressing the model protein were selected for fed-batch culture. Fed-batch culture was carried out in a DASGIP reactor (Eppendorf, Germany) with a final working volume of 0.5 L. The medium and trace element solution were prepared as previously described in Example 7, except that the glycerol concentration in the glycerol batch medium was 45 g per liter. The dissolved oxygen level during culturing was controlled to DO = 30% by the speed of the stirrer (400 to 1200 rpm). The aeration rate was 1 vvm of air, the temperature was controlled to 25 ° C., and the pH setting point of 5.0 was controlled by _{the addition of NH 4} OH (25%). To initiate culture in the bioreactor, 250 mL batch medium was inoculated from the preculture of each methanol-utilizing yeast (P. pastoris) clone with a starting optical concentration (OD600) of 1.0. The batch period with glycerol reached a dry biomass concentration of 25-29 g per liter over about 30 hours. Following the glycerol batch period, restricted glucose fed-batch was performed. Two different fed-batch culture methods: (A) standard fed-batch protocol using a constant feed rate, (B) fed-batch protocol with optimized space yield (“fast fermentation”), glucose feed rate. We compared fed-batch protocols optimized to maximize the volume productivity of fermentation.

For standard cultures, a constant glucose feed rate of 1.25 mL h ^-1 was selected. As a result of maintaining the fed-batch culture for 100 hours (with a total culture time of 126 hours), a final dry biomass concentration of ^{about 90 g L-1 was obtained.} A model-based optimization algorithm (Maurer et al., Microbial Cell Factories, 2006, 5:37) was used for "fast fermentation", where the volume-optimized glucose feed rate F (t) is linear. increasing function: approximated by ^{F (t) [mL h -1} ] = 0.3234mL h -2 × t + 3.3921mL h -1. Maintaining the fed-batch period over t = 33 hours (with a total culture time of 60 hours) resulted in a final dry biomass concentration of ^{approximately 140 g L-1.}

Samples were taken at the end of the batch and at the fed-batch period. Product titers were analyzed with clarified supernatants using the HT low MW protein express reagent kit and the Caliper LabChip GXI system (Perkin Elmer, USA). An alternative scaffold protein purification reference was used as the reference reference for absolute quantification.

FIG. 9 shows the product and biomass production over the total culture time for standard culture (A) and "fast fermentation" (B). By comparison, final product titers of ^{6.4 g L -1} and 4.3 g L ^-1 could be reached after 60 and 126 hours for "fast fermentation" and standard fermentation, respectively. In other words, instead of using the standard feed regimen described, the glucose feed was supplemented upon expression under the pG1-D1240 (SEQ ID NO: 49) promoter as described for "fast fermentation". In a clearly short fermentation time (-66 hours), 1.4 times the titer (corresponding to 1.2 times the culture solution titer) could be found.

Table 1: TFBS identified within the pG1 promoter sequence using MatInspector. The TFBS associated with the carbon source of the target pG1 deletion mutant is shown in bold.

Table 2: TFBS of the pG1 promoter sequence affected in the deletion mutants pG1-Δ1 to Δ12. Sequence analysis was performed using a MatInspector manufactured by Genomatic. The glucose and carbon related TFBS selected for the deletion are shown in bold and the corresponding IDs (1-12) and the deleted location are specified in columns 1 and 2.

Table 3: Location of pG1 TFBS Deletion Mutant and Deletion of TFBS The target TFBS and affected TFBS in the pG1 TFBS deletion mutant (pG1-Δ1 to Δ12) are listed. The target TFBS associated with the carbon source is shown in bold. Detailed information about all TFBSs and deleted TFBSs is presented in Tables 1 and 2, respectively.

Table 4: Primer sequence

Table 5: Relative to the end of fed-batch culture batch and fed-batch of pG1 (here referred to as pG1 # 8) and pG1-x mutant (also referred to here as pG1 mutant) expressing eGFP. Shows eGFP fluorescence. At the end of the batch, the time point was set to 0. Clones expressing eGFP under the control of pG1 (# 8) are subjected to the control of the pG1 deletion mutant (pG1-Δ2), TAT14 mutant (pG1-T16), and duplicate mutant (pG1-D1240). Compared with clones expressing eGFP. Biomass concentration (YDM) in batches and fed-batch was as expected.

Table 6: Promoter intensity compared to pG1 and promoter induction ratio of pG1 mutants by deep well comparative screening. The expression intensity of the pG1-x mutant (induced) is related to the level of eGFP expression obtained for the original pG1 promoter. The induction ratio is calculated from the GFP level in the induced and suppressed states.

The inventions described in the claims at the time of filing are added below.
[1] An isolated and / or artificial pG1-x promoter that is a functional variant of the pG1 promoter regulated by a carbon source of methanol-utilizing yeast (Pichia pastoris) identified by SEQ ID NO: 1. The pG1-x promoter comprises or comprises at least a portion of SEQ ID NO: 1 having a length of at least 293 bp and comprises the following promoter regions:
a) At least one core regulatory region comprising the nucleotide sequences of SEQ ID NO: 2 and SEQ ID NO: 3;
b) A non-core regulatory region, which is an arbitrary region in the pG1-x promoter sequence other than the core regulatory region.
The pG1-x promoter has at least one mutation in any of the promoter regions, at least 80% sequence identity in SEQ ID NO: 2 and SEQ ID NO: 3, and other than SEQ ID NO: 2 or SEQ ID NO: 3. Includes at least 50% sequence identity in any region of the
The pG1-x promoter is characterized by promoter intensity and induction ratio that is the same or increased as compared to the pG1 promoter, in this case.
-The promoter strength is increased at least 1.1 times and / or in the induced state as compared with the pG1 promoter.
The induction ratio is increased by at least 1.1 times as compared to the pG1 promoter.
pG1-x promoter.
[2] The pG1-x promoter according to [1], wherein SEQ ID NO: 2 and / or SEQ ID NO: 3 comprises one or more transcription factor binding sites (TFBS).
[3] The core regulatory region comprises a nucleotide sequence of SEQ ID NO: 4, or a functional variant thereof comprising one or more TFBSs, preferably a functional variant with at least 80% sequence identity. The pG1-x promoter according to [1] or [2].
[4] The core regulatory region is represented by a functional variant thereof comprising SEQ ID NO: 5 or one or more of the TFBSs, preferably a functional variant with at least 80% sequence identity. The pG1-x promoter according to any one of [1] to [3], which is incorporated into the regulatory region.
[5] Any of [1] to [4], wherein the one or more TFBS is a TFBS for any of the transcription factors selected from the group consisting of Rgt1, Cat8-1, and Cat8-2. The pG1-x promoter according to one.
[6] A functional variant of the pG1 promoter containing a deletion of one or more nucleotides at the 5'end of the pG1 sequence, preferably the 3'side region of the pG1 sequence, or the 3'side. The pG1-x promoter according to any one of [1] to [5], which leaves at least 293 nucleotides among the functional variants of the region.
[7] Any one of [1] to [6], wherein the core regulatory region contains a deletion of one or more nucleotides between the nucleotide sequence of SEQ ID NO: 2 and the nucleotide sequence of SEQ ID NO: 3. The pG1-x promoter according to.
[8] The pG1-x promoter according to any one of [1] to [7], which comprises at least two copies of the core or major regulatory region.
[9] The pG1-x promoter according to any one of [1] to [8], comprising at least one or at least two T motifs, identified by any of SEQ ID NOs: 12-29.
[10] The pG1-x promoter according to any one of [1] to [9], wherein the T motif is arranged upstream of the core regulatory region and optionally upstream of the major regulatory region.
[11] The pG1-x promoter according to any one of [1] to [9], wherein the T motif is arranged downstream of the core regulatory region and optionally downstream of the major regulatory region.
[12] The pG1-x promoter according to any one of [1] to [11], which comprises a nucleotide sequence at the 3'end containing at least a part of a translation initiation site.
[13] The pG1-x promoter according to any one of [1] to [12], which has a length of 2000 bp or less.
[14] a) Any of SEQ ID NOs: 37 to 44, preferably SEQ ID NOs: 45 to 76;
b) Any of SEQ ID NOs: 77-80, preferably SEQ ID NOs: 81-112;
c) Any of SEQ ID NOs: 113-114, preferably SEQ ID NOs: 115-130;
d) Any of SEQ ID NOs: 131-132, preferably SEQ ID NOs: 133-148;
e) Any of SEQ ID NOs: 149-150, preferably SEQ ID NOs: 151-166;
f) Any of SEQ ID NOs: 167 to 168, preferably SEQ ID NOs: 169 to 184;
g) Any of SEQ ID NOs: 185 to 186, preferably SEQ ID NOs: 187 to 202;
h) Any of SEQ ID NOs: 203-204, preferably SEQ ID NOs: 205-220;
i) Any of SEQ ID NOs: 221 to 222, preferably SEQ ID NOs: 223 to 238;
j) Any of SEQ ID NOs: 239-240, preferably SEQ ID NOs: 241-256; and
k) SEQ ID NOs: 32-36 or SEQ ID NOs: 257-259;
Containing or consisting of nucleotide sequences selected from the group consisting of any of the following;
Or
l) An isolated and / or artificial pG1-x promoter that is a functional variant of any of the above a) to k).
The pG1-x promoter, which is a functional variant of the pichia pastoris identified by SEQ ID NO: 1, which is a functional variant of the pG1 promoter regulated by a carbon source.
[15] A functional variant of any of the nucleotide sequences, preferably any of the functional variants of SEQ ID NOs: 45-76, characterized by:
a) The sequence contains a deletion of one or more nucleotides at the 5'end of the promoter sequence, preferably among functional variants of the 3'side region of the promoter sequence, or the 3'side region. Being a functional variant of any of the promoter sequences of the pG1-x promoters a) to k) above, leaving at least 293 nucleotides;
b) The sequence comprises one or more TFBSs, preferably the TFBS is a TFBS for any of the transcription factors selected from the group consisting of Rgt1, Cat8-1, and Cat8-2. ;
c) The core regulatory region comprises a nucleotide sequence of SEQ ID NO: 4, or a functional variant thereof comprising one or more TFBSs, preferably a functional variant with at least 80% sequence identity;
d) Incorporate the core regulatory region into a major regulatory region represented by SEQ ID NO: 5, or a functional variant thereof comprising said TFBS, preferably a functional variant with at least 80% sequence identity. Being;
e) The core regulatory region comprises a deletion of one or more nucleotides between the nucleotide sequence of SEQ ID NO: 2 and the nucleotide sequence of SEQ ID NO: 3;
f) The sequence comprises at least two copies of the core regulatory region or the major regulatory region;
g) The sequence comprises at least one or at least two T-motifs identified by any of SEQ ID NOs: 12-29, preferably said T-motif upstream or downstream of said core regulatory region. Arranged and optionally located upstream or downstream of said major regulatory region;
h) The sequence comprises a 3'end nucleotide sequence containing at least a portion of the translation initiation site;
i) The sequence is extended to a length of 2000 bp or less.
The pG1-x promoter according to [14], which is a functional variant characterized by one or more of the above.
[16] An isolated pG1-x promoter nucleic acid containing the pG1-x promoter according to any one of [1] to [15], or a nucleic acid containing a complementary sequence.
[17] The pG1 according to [16], which is operably linked to a nucleotide sequence encoding a protein of interest (POI) and does not naturally associate with the nucleotide sequence encoding the POI. -X promoter nucleic acid.
[18] Further comprising a nucleotide sequence encoding a signal peptide that allows the secretion of the POI, preferably the nucleotide sequence encoding the signal peptide is flanked by the 5'end of the nucleotide sequence encoding the POI. The nucleic acid according to [17].
[19] An expression construct containing the nucleic acid according to any one of [16] to [18], preferably a self-replicating vector or self-replicating plasmid, or a vector or plasmid that is integrated into the chromosomal DNA of a host cell. An expression construct.
[20] Recombinant host cells containing the expression construct according to [19], preferably eukaryotic cells, more preferably yeast cells or filamentous fungal cells, more preferably Saccharomyces or Pichia. Recombinant host cells, which are yeast cells of the genus.
[21] A method for producing POI by culturing the recombinant host cell line according to [20].
a) The step of cultivating the cell line under the condition of expressing the POI, and
b) With the step of collecting the POI
How to include.
[22] Culture
a) Suppression of the pG1-x promoter by using a basal carbon source, the first step followed by
b) With the second step of inducing the production of the POI by releasing the suppression of the pG1-x promoter by not using the supplemental carbon source or by using a limited amount of the supplementary carbon source.
21. The method according to [21].
[23] The second step b) supplies the supplementary carbon source in a growth limiting amount to increase the specific growth rate to 0.001 h. ^-1 ~ 0.2h ^-1 , Preferably 0.005h ^-1 ~ 0.15h ^-1 The method according to [22], wherein the feed medium is used, which is kept within the range of.
[24] a) The basal carbon source is selected from the group consisting of glucose, glycerol, ethanol, mixtures thereof, and complex nutritional materials;
b) The supplemental carbon source is a hexose, such as glucose, fructose, galactose or mannose, a disaccharide, such as saccharose, alcohol, such as glycerol or ethanol, or a mixture of any of the above.
The method according to [22] or [23].
[25] The culture is carried out in a bioreactor in a batch period as the first step, followed by a fed-batch period or a continuous culture period as the second step, [22] to [ 24] The method according to any one of.
[26] The method according to [25], wherein the batch period is carried out until the cell line consumes the basic carbon source.
[27] The method according to [25] or [26], wherein the batch period is characterized by a sustained decrease in oxygen partial pressure (pO2) signal, and the end point of the batch period is characterized by an increase in pO2.
[28] The method of [27], wherein during the batch period, the pO2 is reduced to less than 65% or low saturation, followed by an increase or high saturation of greater than 65% at the end of the batch.
[29] The method according to any one of [25] to [28], wherein the batch period is carried out for about 20 to 36 hours.
[30] The batch period is carried out with 45 g of glycerol in 1 L of batch medium and the culture is carried out at 25 ° C. for about 27-30 hours or at 30 ° C. for about 23-36 hours. The method according to any one of [25] to [29].
[31] The culture during the fed-batch period is carried out for about 15 to 80 hours, about 15 to 70 hours, about 15 to 60 hours, about 15 to 50 hours, about 15 to 45 hours, about 15 to 40 hours, about 15 hours. ~ 35 hours, about 15-30 hours, about 15-35 hours, about 15-25 hours, or about 15-20 hours; preferably over any of about 20-40 hours, [25] ~ The method according to any one of [30].
[32] The culture during the fed-batch period was carried out for about 80 hours, about 70 hours, about 60 hours, about 55 hours, about 50 hours, about 45 hours, about 40 hours, about 35 hours, about 30 hours, about 25. The method according to any one of [25] to [31], which is carried out over an hour, about 20 hours, or about 15 hours.
[33] The second step b) supplies a growth limiting amount of the supplemental carbon source to increase the specific growth rate to 0.04 h. ^-1 ~ 0.2h ^-1 The method according to any one of [25] to [32], wherein a feed medium kept within the range of is used in the fed-batch period.
[34] Approximately 30 mg (L h) ^-1 The method according to any one of [25] to [33], which achieves the air-time yield of.
[35] The method according to any one of [34], wherein the culture during the fed-batch period is carried out for about 30 hours.
[36] The recombinant host cell is a yeast of either the genus Saccharomyces or the genus Pichia or the genus Komagataella, preferably the methanol-utilizing yeast (Pichia pastoris) or Komagataera pastoris ( The method according to any one of [25] to [35], which is Komagataella pastoris).
[37] The pG1-x promoter may be any one of [25] to [36], which is any one of SEQ ID NOs: 37 to 44, preferably any of SEQ ID NOs: 45 to 76. The method described.
[38] The method of [37], wherein the pG1-x promoter is characterized by SEQ ID NO: 39, preferably SEQ ID NO: 49.
[39] The method according to any one of [21] to [38], wherein the POI is produced at a transcription rate of at least 15% as compared to the native pGAP promoter of the cell.
[40] The POI is preferably an antibody or fragment thereof, enzymes and peptides, protein antibiotics, toxin fusion proteins, carbohydrate-protein conjugates, structural proteins, regulatory proteins, vaccines and vaccine-like proteins or particles, processing enzymes, The method according to any one of [21] to [39], which is a heterologous protein selected from therapeutic proteins including growth factors, hormones, and cytokines, or metabolites of POI.

Claims (24)

An isolated and / or artificial pG1-x promoter, the pG1-x promoter is one of the following promoter regions:
a) At least two core regulatory promoter regions, wherein each core regulatory promoter region comprises the nucleotide sequences of SEQ ID NO: 2 and SEQ ID NO: 3, and the core regulatory promoter region corresponds within the nucleotide sequence of SEQ ID NO: 1. Has at least 90% sequence identity with the region containing the nucleotide sequence of SEQ ID NO: 4; and b) in any region within the pG1-x promoter sequence other than the at least two core-regulating promoter regions. A non-core regulatory promoter region, wherein said non-core regulatory promoter region has at least 50% sequence identity with the corresponding region within the nucleotide sequence of SEQ ID NO: 1.
Including
The pG1-x promoter contains at least a portion of 293bp to have a 2 93 bp portion and at least 90% sequence identity pG1 promoter of SEQ ID NO: 1, wherein the portion of 293bp of the pG1 promoter Containing positions 639-756 of the nucleotide sequence of SEQ ID NO: 1;
The pG1-x promoter increases promoter strength by at least 1.1-fold in the inducible state as compared to the pG1 promoter and / or induces an increase of at least 1.1-fold as compared to the pG1 promoter. Characterized by an increase in the ratio,
pG1-x promoter. The pG1-x promoter according to claim 1, wherein the nucleotide sequence of SEQ ID NO: 2 and / or SEQ ID NO: 3 comprises one or more transcription factor binding sites (TFBS). At least one of the core regulatory promoter regions comprises (i) the nucleotide sequence of SEQ ID NO: 4 or (ii) the nucleotide sequences of SEQ ID NO: 2 and SEQ ID NO: 3 and is at least 90 % sequence identical to the nucleotide sequence of SEQ ID NO: 4. The pG1-x promoter according to claim 1, wherein the core regulatory promoter region comprises one or more TFBSs comprising a functional variant of the nucleotide sequence of SEQ ID NO: 4 having sex. At least one of the core regulatory promoter regions comprises (i) the nucleotide sequence of SEQ ID NO: 5 or (ii) the nucleotide sequences of SEQ ID NO: 2 and SEQ ID NO: 3 and is at least 80% sequence identical to the nucleotide sequence of SEQ ID NO: 5. The pG1-x promoter according to claim 1, wherein the core regulatory promoter region comprises one or more TFBSs incorporated into a major regulatory region comprising a functional variant of the nucleotide sequence of SEQ ID NO: 5 having sex. Claimed to include a TFBS for a transcription factor selected from the group consisting of glucose transporter transcriptional regulator (Rgt1), zinc cluster transcriptional activator 1 (Cat8-1), and zinc cluster transcriptional activator 2 (Cat8-2). Item 1. The pG1-x promoter according to Item 1. At least one of the core regulatory promoter regions comprises the nucleotide sequences of SEQ ID NO: 2 and SEQ ID NO: 3, with one or more nucleotide deletions between the nucleotide sequence of SEQ ID NO: 2 and the nucleotide sequence of SEQ ID NO: 3. The pG1-x promoter according to claim 1, which comprises. The pG1-x promoter according to claim 4, which comprises at least two copies of the major regulatory region. The pG1-x promoter according to claim 1, comprising at least one or at least two thymine (T) motifs identified by any one of the nucleotide sequences of SEQ ID NOs: 12-29. The pG1-x promoter according to claim 8, wherein the T motif is located upstream of the at least one or both core regulatory promoter regions. The pG1-x promoter according to claim 8, wherein the T motif is located downstream of the at least one or both core regulatory promoter regions. a) Any of SEQ ID NOs: 37-44 or SEQ ID NOs: 45-76;
b) Either SEQ ID NO: 77-80 or SEQ ID NOs: 81-112;
c) Either SEQ ID NO: 113-114 or SEQ ID NO: 115-130;
d) any of SEQ ID NOs: 131-132, or SEQ ID NOs: 133-148; or e) any of SEQ ID NOs: 185-186, or SEQ ID NOs: 187-202;
f) A nucleotide sequence selected from the group consisting of a nucleotide sequence having at least 90% sequence identity with any of the above and containing the nucleotide sequences of SEQ ID NO: 2 and SEQ ID NO: 3 which are core regulatory promoter regions. or an isolated and / or artificial pG1-x promoter that Do these include,
The isolated and / or artificial pG1-x promoter increases promoter strength by at least 1.1-fold in the induced state as compared to the pG1 promoter represented by SEQ ID NO: 1 and / or with the pG1 promoter. It is characterized by an increase in the inducer ratio, which increases by at least 1.1 times in comparison.
pG1-x promoter . The nucleotide sequence has at least 90% sequence identity with any of the nucleotide sequences of SEQ ID NOs: 45-76 and:
a) The sequence comprises one or more TFBSs, the one or more TFBS being a TFBS for any of the transcription factors selected from the group consisting of Rgt1, Cat8-1, and Cat8-2. thing;
b) A functional variant in which at least one of the core regulatory promoter regions has at least 80% sequence identity with the nucleotide sequence of SEQ ID NO: 4, including the nucleotide sequence of SEQ ID NO: 4, or one or more TFBSs. Include;
c) A functional variant in which at least one of the core regulatory promoter regions has at least 80% sequence identity with the nucleotide sequence of SEQ ID NO: 5, including the nucleotide sequence of SEQ ID NO: 5, or one or more TFBSs. Incorporated into the major regulatory region containing; d) At least one of the core regulatory promoter regions comprises the nucleotide sequences of SEQ ID NO: 2 and SEQ ID NO: 3, and the nucleotide sequence of SEQ ID NO: 2 and the nucleotide sequence of SEQ ID NO: 3 Includes deletion of one or more nucleotides between;
e) The sequence comprises at least two major regulatory regions comprising the nucleotide sequence of SEQ ID NO: 5 or its functional variant having at least 80% sequence identity with the nucleotide sequence of SEQ ID NO: 5;
f) The sequence comprises at least one or at least two T motifs identified by any one of the nucleotide sequences of SEQ ID NOs: 12-29;
g) The sequence comprises a 3'-terminal nucleotide sequence containing at least a portion of the translation initiation site;
h) The pG1-x promoter according to claim 11, wherein the sequence comprises one or more of having a length of up to 2000 bp. An isolated pG1-x promoter nucleic acid containing the pG1-x promoter according to claim 1, or a nucleic acid containing a complementary sequence thereof. The pG1-x promoter nucleic acid according to claim 13, which is operably linked to a nucleotide sequence encoding a protein of interest (POI) and does not naturally associate with the nucleotide sequence encoding the POI. .. An expression construct comprising the nucleic acid according to claim 13. A recombinant host cell comprising the expression construct according to claim 15. A method for producing POI by culturing the recombinant host cell line according to claim 16.
a) The step of cultivating the cell line under the condition of expressing the POI, and
b) A method including a step of collecting the POI. Culture
a) First step of suppressing the pG1-x promoter by using a basal carbon source and subsequent b) No supplemental carbon source or a limited amount of supplemental carbon source The method according to claim 17, further comprising a second step of releasing the suppression of the pG1-x promoter and inducing the production of the POI. The method of claim 18, wherein the pG1-x promoter is any of the nucleotide sequences of SEQ ID NOs: 37-44. 19. The method of claim 19, wherein the pG1-x promoter is characterized by the nucleotide sequence of SEQ ID NO: 39. The recombinant host cell according to claim 16, wherein the host cell is a eukaryotic cell. The recombinant host cell according to claim 21, wherein the eukaryotic cell is a yeast cell or a filamentous fungal cell. The recombinant host cell according to claim 21, wherein the eukaryotic cell is a yeast cell of the genus Saccharomyces or Pichia. The pG1-x promoter according to claim 1, wherein the at least two core regulatory promoter regions are the same.